Degradation of cyclin-dependent kinase 8 (CDK8) by conjugation of CDK8 inhibitors with E3 ligase ligand and methods of use

ABSTRACT

The present application provides bifunctional compounds of Formula (Ia) or (Ib): 
                         
or a pharmaceutically acceptable salt, hydrate, solvate, prodrug, stereoisomer, or tautomer thereof, which act as protein degradation inducing moieties for cyclin-dependent kinase 8 (CDK8). The present application also relates to methods for the targeted degradation of CDK8 through the use of the bifunctional compounds that link a ubiquitin ligase-binding moiety to a ligand that is capable of binding to CDK8 which can be utilized in the treatment of disorders modulated by CDK8.

RELATED APPLICATION

This application is a continuation of U.S. application Ser. No.17/083,940, filed on Oct. 29, 2020, which is a continuation of U.S.application Ser. No. 16/094,105, filed on Oct. 16, 2018 (now U.S. Pat.No. 10,865,205), which is a U.S. National Phase application, filed underU.S.C. § 371, of International Application No. PCT/US2017/028948, filedon Apr. 21, 2017, which claims priority to, and the benefit of, U.S.Application No. 62/326,584, filed on Apr. 22, 2016, the entire contentsof each of which are incorporated herein by reference.

GOVERNMENT SUPPORT

This invention was made with government support under grant number R01CA179483 awarded by The National Institutes of Health. The governmenthas certain rights in the invention.

BACKGROUND

Ubiquitin-Proteasome Pathway (UPP) is a critical pathway that regulatesproteins and degrades misfolded or abnormal proteins. UPP is central tomultiple cellular processes, and if defective or imbalanced, leads topathogenesis of a variety of diseases. The covalent attachment ofubiquitin to specific protein substrates is achieved through the actionof E3 ubiquitin ligases. These ligases comprise over 500 differentproteins and are categorized into multiple classes defined by thestructural element of their E3 functional activity. For example,cereblon (CRBN) interacts with damaged DNA binding protein 1 and formsan E3 ubiquitin ligase complex with Cullin 4 in which the proteinsrecognized by CRBN are ubiquitinated and degraded by proteasomes.Various immunomodulatory drugs (IMiDs), e.g. thalidomide andlenalidomide, binds to CRBN and modulates CRBN's role in theubiquitination and degradation of protein factors involved inmaintaining regular cellular function.

Bifunctional compounds composed of a target protein-binding moiety andan E3 ubiquitin ligase-binding moiety have been shown to induceproteasome-mediated degradation of selected proteins. These drug-likemolecules offer the possibility of temporal control over proteinexpression, and could be useful as biochemical reagents for thetreatment of diseases.

Cyclin-dependent kinase is a kinase family integrating multiplesignaling pathways to control either cell cycle or gene transcription.CDK1, 2, 4 and 6 are the critical enzymes that drive cell cycletransition. For example, CDK1 is a key determinant of mitoticprogression, CDK2 regulates DNA replication in S phase, and CDK4/6drives the cell cycle from G0 or G1 to S phase by phosphorylation on Rbprotein to activate expression of genes involved in cell cycle control.CDK7, 9 and 12 are known enzymes that regulate the transcription insteadof directly promoting cell cycles. CDK7 is the enzymatic component ofTFIIH complex which is responsible for regulating transcriptioninitiation, and CDK9 and CDK12 regulate transcription elongation andprocessing.

Deregulation of CDKs has been shown to have a significant impact on thecell state and is frequently identified as oncogenic. Numerous selectiveor pan-CDK small molecule inhibitors have been identified, however, mostof the known inhibitors have failed in clinic trials due to the lack ofhigh systemic drug concentration. More recently, the development of aCDK7 covalent inhibitor, THZ1, has demonstrated that irreversiblebinders are superior to reversible CDK binders.

Alternative strategies to inhibit cyclin-dependent kinases, such asCDK8, are needed. At present, suitable compounds with alternativemechanisms of action targeting CDK8 are not available. The presentapplication addresses the need.

SUMMARY

The present application relates to novel bifunctional compounds, whichfunction to recruit targeted proteins to E3 ubiquitin ligase fordegradation, and methods of preparation and uses thereof. Thebifunctional compound is of Formula X:TL-L-D  (X),wherein:

-   -   the Targeting Ligand (TL) is capable of binding to a targeted        protein, such as a cyclin-dependent kinase (e.g., CDK8);    -   the Linker (L) is a group that covalently binds to the Targeting        Ligand and the Degron; and    -   the Degron (D) is capable of binding to a ubiquitin ligase, such        as an E3 ubiquitin ligase (e.g., cereblon).

The present application also relates to targeted degradation of proteinsthrough the use of bifunctional compounds, including bifunctionalcompounds that link an E3 ubiquitin ligase-binding moiety to a ligandthat binds the targeted proteins.

The present application also relates to a bifunctional compound ofFormula Ia or Ib:

or an enantiomer, diastereomer, stereoisomer, or pharmaceuticallyacceptable salt thereof,wherein:

-   -   R₁, R₂, R₃, R₅, R₆, R₇, A, A′, B′, X, X₂, X₃, X₅, n, n′, o, o′,        t and r′ are each as defined herein;    -   the Linker is a group that covalently binds to

-   -    in Formula (Ia) or

-   -    in Formula (Ib) and the Degron;    -   the Degron is capable of binding to a ubiquitin ligase, such as        an E3 ubiquitin ligase (e.g., cereblon); and    -   the Targeting Ligand is capable of binding to a targeted        protein, such as CDK8.

The present application further relates to a Degron of Formula D1:

or an enantiomer, diastereomer, or stereoisomer thereof, wherein Y, Z,R₁₃, R₁₄, R₁₅, R₁₆, v, and q are each as defined herein.

The present application further relates to a Linker of Formula L0:

or an enantiomer, diastereomer, or stereoisomer thereof, wherein p1, p2,p3, W, Q, and Z₁ are each as defined herein, the Linker is covalentlybonded to a Degron via the

next to Q, and covalently bonded to a Targeting Ligand via the

next to Z₁.

The present application also relates to a pharmaceutical compositioncomprising a therapeutically effective amount of a bifunctional compoundof the application, or an enantiomer, diastereomer, stereoisomer, orpharmaceutically acceptable salt thereof, and a pharmaceuticallyacceptable carrier.

Another aspect of the present application relates to a method ofinhibiting a kinase (e.g., CDK8). The method comprises administering toa subject in need thereof an effective amount of a bifunctional compoundof the application, or a pharmaceutically acceptable salt, hydrate,solvate, prodrug, stereoisomer, or tautomer thereof, or a pharmaceuticalcomposition of the application.

Another aspect of the present application relates to a method ofmodulating (e.g., decreasing) the amount of a kinase (e.g., CDK8). Themethod comprises administering to a subject in need thereof atherapeutically effective amount of a bifunctional compound of theapplication, or a pharmaceutically acceptable salt, hydrate, solvate,prodrug, stereoisomer, or tautomer thereof, or a pharmaceuticalcomposition of the application.

Another aspect of the present application relates to a method oftreating or preventing a disease (e.g., a disease in which CDK8 plays arole). The method comprises administering to a subject in need thereofan effective amount of a bifunctional compound of the application, or apharmaceutically acceptable salt, hydrate, solvate, prodrug,stereoisomer, or tautomer thereof, or a pharmaceutical composition ofthe application. In one aspect, the disease is a kinase (e.g., CDK8)mediated disorder. In one aspect, the disease is a proliferative disease(e.g., a proliferative disease in which CDK8 plays a role).

Another aspect of the present application relates to a method oftreating or preventing cancer in a subject, wherein the cancer cellcomprises an activated CDK8 or wherein the subject is identified asbeing in need of CDK8 inhibition for the treatment or prevention ofcancer. The method comprises administering to the subject an effectiveamount of a bifunctional compound of the application, or apharmaceutically acceptable salt, hydrate, solvate, prodrug,stereoisomer, or tautomer thereof, or a pharmaceutical composition ofthe application.

Another aspect of the present application relates to a kit comprising abifunctional compound capable of inhibiting CDK8 activity, selected froma bifunctional compound of the application, or a pharmaceuticallyacceptable salt, hydrate, solvate, prodrug, stereoisomer, or tautomerthereof.

Another aspect of the present application relates to a kit comprising abifunctional compound capable of modulating (e.g., decreasing) theamount of CDK8, selected from a bifunctional compound of theapplication, or a pharmaceutically acceptable salt, hydrate, solvate,prodrug, stereoisomer, or tautomer thereof.

Another aspect of the present application relates to a bifunctionalcompound of the application, or a pharmaceutically acceptable salt,hydrate, solvate, prodrug, stereoisomer, or tautomer thereof, or apharmaceutical composition of the application, for use in themanufacture of a medicament for inhibiting a kinase (e.g., CDK8) or formodulating (e.g., decreasing) the amount of a kinase (e.g., CDK8).

Another aspect of the present application relates to a bifunctionalcompound of the application, or a pharmaceutically acceptable salt,hydrate, solvate, prodrug, stereoisomer, or tautomer thereof, or apharmaceutical composition of the application, for use in themanufacture of a medicament for treating or preventing a disease (e.g.,a disease in which CDK8 plays a role). In one aspect, the disease is akinase (e.g., CDK8) mediated disorder. In one aspect, the disease is aproliferative disease (e.g., a proliferative disease in which CDK8 playsa role).

Another aspect of the present application relates to a bifunctionalcompound of the application, or a pharmaceutically acceptable salt,hydrate, solvate, prodrug, stereoisomer, or tautomer thereof, or apharmaceutical composition of the application, for use in themanufacture of a medicament for treating or preventing cancer in asubject, wherein the cancer cell comprises an activated CDK8 or whereinthe subject is identified as being in need of CDK8 inhibition for thetreatment or prevention of cancer.

Another aspect of the present application relates to a bifunctionalcompound of the application, or a pharmaceutically acceptable salt,hydrate, solvate, prodrug, stereoisomer, or tautomer thereof, or apharmaceutical composition of the application, for use in inhibiting akinase (e.g., CDK8) or modulating (e.g., decreasing) the amount of akinase (e.g., CDK8).

Another aspect of the present application relates to a bifunctionalcompound of the application, or a pharmaceutically acceptable salt,hydrate, solvate, prodrug, stereoisomer, or tautomer thereof, or apharmaceutical composition of the application, for use in treating orpreventing a disease (e.g., a disease in which CDK8 plays a role). Inone aspect, the disease is a kinase (e.g., CDK8) mediated disorder. Inone aspect, the disease is a proliferative disease (e.g., aproliferative disease in which CDK8 plays a role).

Another aspect of the present application relates to a bifunctionalcompound of the application, or a pharmaceutically acceptable salt,hydrate, solvate, prodrug, stereoisomer, or tautomer thereof, or apharmaceutical composition of the application, for use in treating orpreventing cancer in a subject, wherein the cancer cell comprises anactivated CDK8 or wherein the subject is identified as being in need ofCDK8 inhibition for the treatment or prevention of cancer.

The present application provides inhibitors of CDK8 that are therapeuticagents in the treatment or prevention of diseases such as cancer andmetastasis.

The present application further provides compounds and compositions withan improved efficacy and/or safety profile relative to known CDK8inhibitors. The present application also provides agents with novelmechanisms of action toward CDK8 kinases in the treatment of varioustypes of diseases including cancer and metastasis.

The compounds and methods of the present application address unmet needsin the treatment of diseases or disorders in which pathogenic oroncogenic endogenous proteins (e.g., CDK8) play a role, such as cancer.

The details of the disclosure are set forth in the accompanyingdescription below. Although methods and materials similar or equivalentto those described herein can be used in the practice or testing of thepresent application, illustrative methods and materials are nowdescribed. In the case of conflict, the present specification, includingdefinitions, will control. In addition, the materials, methods, andexamples are illustrative only and are not intended to be limiting.Other features, objects, and advantages of the disclosure will beapparent from the description and from the claims. In the specificationand the appended claims, the singular forms also include the pluralunless the context clearly dictates otherwise. Unless defined otherwise,all technical and scientific terms used herein have the same meaning ascommonly understood by one of ordinary skill in the art to which thisdisclosure belongs.

The contents of all references (including literature references, issuedpatents, published patent applications, and co-pending patentapplications) cited throughout this application are hereby expresslyincorporated herein in their entireties by reference. The referencescited herein are not admitted to be prior art to the application.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1A is a graph showing anti-proliferative effects of Compound I-1,Compound I-2, and Compound I-3 in wild-type (WT) or cereblon knockout(CRBN−/−) cells at various concentrations. Antiproliferative effectswere assessed 72 hours after treatment.

FIG. 1B is a graph showing anti-proliferative effects of Compound I-6,Compound I-7, and Compound I-8 in wild-type (WT) or cereblon knockout(CRBN−/−) cells at various concentrations. Antiproliferative effectswere assessed 72 hours after treatment.

FIG. 2 is a western blot showing the levels of CDK8, pS727-STAT1, andactin in MC38 colorectal cancer cells treated with 1 μM Compound I-1,Compound I-2, Compound I-3, or DMSO, alone or in combination with 10 nMbortezomib for 6 hours. Compound I-3 efficiently degraded CDK8 andlowered the level of phosphorylation of the known CDK8 substrateSer727-STAT1. Inhibiting the proteasome with bortezomib rescued both thedegradation of CDK8 and the reduction in the level of pSer727-STAT1.

FIG. 3A and FIG. 3B are western blots showing the levels of mTOR, CDK8,pS727-STAT1, and actin in wild-type (WT) (FIG. 3A) or cereblon knockout(CRBN−/−) (FIG. 3B) Molt4 cells treated with DMSO or 1 μM Compound I-1,I-2, I-3, I-4, I-5, I-6, I-7, or I-8 for 6 hours. Compounds I-3 and I-6efficiently degraded CDK8 and blocked phosphorylation of Ser727-STAT1 inWT Molt4 cells. CDK8 was not degraded in CRBN−/− Molt4 cells.

FIG. 4A is a western blot showing the levels of CDK8 and actin in cellstreated with the indicated concentrations of Compound I-3 or DMSO.

FIG. 4B is a western blot showing the levels of CDK8 and tubulin inMolt4 cells treated for 4 hours with the indicated concentrations ofCompound I-6 or DMSO.

FIG. 4C is a western blot showing the levels of CDK8, pS727-STAT1, andactin in Jurkat cells treated with the indicated concentrations ofCompound I-9 or DMSO.

DETAILED DESCRIPTION

Compounds of the Application

The present application relates to bifunctional compounds having utilityas modulators of ubiquitination and proteosomal degradation of targetedproteins, especially compounds comprising a moiety capable of binding toa polypeptide or a protein that is degraded and/or otherwise inhibitedby the bifunctional compounds of the present application. In particular,the present application is directed to compounds which contain a moiety,e.g., a small molecule moiety (i.e., having a molecular weight of below2,000, 1,000, 500, or 200 Daltons), such as a thalidomide-like moiety,which is capable of binding to an E3 ubiquitin ligase, such as cereblon,and a ligand that is capable of binding to a target protein, in such away that the target protein is placed in proximity to the ubiquitinligase to effect degradation (and/or inhibition) of that protein.

In one embodiment, the present application provides a bifunctionalcompound of Formula X:TL-L-D  (X),wherein:

-   -   the Targeting Ligand (TL) is capable of binding to a targeted        protein, such as CDK8;    -   the Linker (L) is a group that covalently binds to the Targeting        Ligand and the Degron; and    -   the Degron (D) is capable of binding to a ubiquitin ligase, such        as an E3 ubiquitin ligase (e.g., cereblon).

In one embodiment, the present application provides a compound ofFormula Ia:

or an enantiomer, diastereomer, stereoisomer, or pharmaceuticallyacceptable salt thereof,wherein:

-   -   R₁, R₂, R₃, A, X, X₂, X₃, n, o, and t are each as defined        herein;    -   the Linker is a group that covalently binds to

-   -    and the Degron;    -   the Degron is capable of binding to a ubiquitin ligase, such as        an E3 ubiquitin ligase (e.g., cereblon); and    -   the Targeting Ligand is capable of binding to a targeted        protein, such as CDK8.

In one embodiment, the present application provides a compound ofFormula Ib:

-   -   or an enantiomer, diastereomer, stereoisomer, or        pharmaceutically acceptable salt thereof,        wherein:    -   R₅, R₆, R₇, A′, B′, X₅, n′, o′, and r′ are each as defined        herein;    -   the Linker is a group that covalently binds to

-   -    and the Degron;    -   the Degron is capable of binding to a ubiquitin ligase, such as        an E3 ubiquitin ligase (e.g., cereblon); and    -   the Targeting Ligand is capable of binding to a targeted        protein, such as CDK8.

The present application further relates to a Degron of Formula D1:

or an enantiomer, diastereomer, or stereoisomer thereof, wherein Y, Z,R₁₃, R₁₄, R₁₅, R₁₆, q, and v are each as defined herein.

The present application further relates to a Linker of Formula L0:

or an enantiomer, diastereomer, or stereoisomer thereof, wherein p1, p2,p3, W, Q, and Z₁ are each as defined herein, the Linker is covalentlybonded to a Degron via the

next to Q, and covalently bonded to the Targeting Ligand via the

next to Z₁.Targeting Ligand

Targeting Ligand (TL) (or target protein moiety or target protein ligandor ligand) is a small molecule which is capable of binding to a targetprotein of interest, such CDK8.

In one embodiment, a Targeting Ligand is a compound of Formula TL-I:

or an enantiomer, diastereomer, stereoisomer, or pharmaceuticallyacceptable salt thereof,wherein:

-   -   A is

-   -   X is N or CH;    -   X₂ is N or CH;    -   X₃ is N or CH;    -   X₄ is N or CH;    -   each R₁ is independently (C₁-C₄) alkyl or (C₁-C₄) haloalkyl;    -   each R₂ is independently (C₁-C₄) alkyl, (C₁-C₄) haloalkyl,        (C₁-C₄) alkoxy, (C₁-C₄) haloalkoxy, halogen, OH, or NH₂;    -   each R₃ is independently (C₁-C₄) alkyl, (C₁-C₄) haloalkyl,        (C₁-C₄) alkoxy, (C₁-C₄) haloalkoxy, halogen, OH, or NH₂;    -   each R₄ is independently (C₁-C₄) alkyl, (C₁-C₄) haloalkyl,        (C₁-C₄) alkoxy, (C₁-C₄) haloalkoxy, halogen, OH, or NH₂; and    -   t, n, o, and r are each independently 0, 1, 2, or 3;        wherein the Targeting Ligand is bonded to the Linker via the

next to

In some embodiments, A is

In other embodiments, A is

In other embodiments, A is

In other embodiments, A is

In some embodiments, X is N. In other embodiments, X is CH.

In some embodiments, each R₁ is independently methyl, ethyl, propyl, ori-propyl. In other embodiments, each R₁ is independently is methyl orethyl. In other embodiments, each R₁ is independently is methyl. Inother embodiments, each R₁ is independently is (C₁-C₄) haloalkyl (e.g.,CF₃, CHF₂, CH₂CF₃, or CF₂CF₃).

In some embodiments, each R₂ is independently (C₁-C₃) alkyl, (C₁-C₃)haloalkyl, (C₁-C₃) alkoxy, (C₁-C₃) haloalkoxy, halogen, OH, or NH₂. Inother embodiments, each R₂ is independently (C₁-C₃) alkyl, (C₁-C₃)haloalkyl, halogen, OH, or NH₂. In other embodiments, each R₂ isindependently halogen, OH, or NH₂. In other embodiments, each R₂ isindependently (C₁-C₃) alkyl or (C₁-C₃) haloalkyl. In other embodiments,each R₂ is independently (C₁-C₃) alkyl, (C₁-C₃) haloalkyl, (C₁-C₃)alkoxy, or (C₁-C₃) haloalkoxy. In other embodiments, each R₂ isindependently (C₁-C₃) alkyl or halogen.

In some embodiments, each R₃ is independently (C₁-C₃) alkyl, (C₁-C₃)haloalkyl, (C₁-C₃) alkoxy, (C₁-C₃) haloalkoxy, halogen, OH, or NH₂. Inother embodiments, each R₃ is independently (C₁-C₃) alkyl, (C₁-C₃)haloalkyl, halogen, OH, or NH₂. In other embodiments, each R₃ isindependently halogen, OH, or NH₂. In other embodiments, each R₃ isindependently (C₁-C₃) alkyl or (C₁-C₃) haloalkyl. In other embodiments,each R₃ is independently (C₁-C₃) alkyl, (C₁-C₃) haloalkyl, (C₁-C₃)alkoxy, or (C₁-C₃) haloalkoxy. In other embodiments, each R₃ isindependently (C₁-C₃) alkyl or halogen.

In some embodiments, each R₄ is independently (C₁-C₃) alkyl, (C₁-C₃)haloalkyl, (C₁-C₃) alkoxy, (C₁-C₃) haloalkoxy, halogen, OH, or NH₂. Inother embodiments, each R₄ is independently (C₁-C₃) alkyl, (C₁-C₃)haloalkyl, halogen, OH, or NH₂. In other embodiments, each R₄ isindependently halogen, OH, or NH₂. In other embodiments, each R₄ isindependently (C₁-C₃) alkyl or (C₁-C₃) haloalkyl. In other embodiments,each R₄ is independently (C₁-C₃) alkyl, (C₁-C₃) haloalkyl, (C₁-C₃)alkoxy, or (C₁-C₃) haloalkoxy. In other embodiments, each R₄ isindependently (C₁-C₃) alkyl or halogen. In other embodiments, each R₄ isindependently methyl, ethyl, propyl, trifluoromethyl, or difluoromethyl.In other embodiments, each R₄ is independently trifluoromethyl ordifluoromethyl. In other embodiments, at least one R₄ istrifluoromethyl.

In some embodiments, t is 0. In other embodiments, t is 1. In otherembodiments, t is 2. In other embodiments, t is 3. In other embodiments,t is 0 or 1. In other embodiments, t is 1 or 2. In other embodiments, tis 0, 1 or 2. In other embodiments, t is 1, 2 or 3.

In some embodiments, n is 0. In other embodiments, n is 1. In otherembodiments, n is 2. In other embodiments, n is 3. In other embodiments,n is 0 or 1. In other embodiments, n is 1 or 2. In other embodiments, nis 0, 1 or 2. In other embodiments, n is 1, 2 or 3.

In some embodiments, o is 0. In other embodiments, o is 1. In otherembodiments, o is 2. In other embodiments, o is 3. In other embodiments,o is 0 or 1. In other embodiments, o is 1 or 2. In other embodiments, ois 0, 1 or 2. In other embodiments, o is 1, 2 or 3.

In some embodiments, r is 0. In other embodiments, r is 1. In otherembodiments, r is 2. In other embodiments, r is 3. In other embodiments,r is 0 or 1. In other embodiments, r is 1 or 2. In other embodiments, ris 0, 1 or 2. In other embodiments, r is 1, 2 or 3.

Any of the groups described herein for any of A, X, X₂, X₃, X₄, R₁, R₂,R₃, R₄, n, o, r, and t can be combined with any of the groups describedherein for one or more of the remainder of A, X, X₂, X₃, X₄, R₁, R₂, R₃,R₄, n, o, r, and t, and may further be combined with any of the groupsdescribed herein for the Linker.

For a Targeting Ligand of Formula TL-I:

-   -   (1) In one embodiment, X is N and A is

-   -   (2) In one embodiment, X is N and A is

-   -   (3) In one embodiment, X is N and X₂ is N.    -   (4) In one embodiment, X is N and X₃ is N.    -   (5) In one embodiment, X is N, X₂ is N, and X₃ is N.    -   (6) In one embodiment, X is N, X₂ is N, X₃ is N, and A is

-   -   (7) In one embodiment, X is N, X₂ is N, X₃ is N, and A is

-   -   (8) In one embodiment, X is N, X₂ is N, X₃ is N, A is

-   -    and o is 0.    -   (9) In one embodiment, X is N, X₂ is N, X₃ is N, A is

-   -    and n is 0.    -   (10) In one embodiment, X is N, X₂ is N, X₃ is N, A is

-   -    and t is 0.    -   (11) In one embodiment, X is N, X₂ is N, X₃ is N, A is

-   -    and r is 1.    -   (12) In one embodiment, X is N, X₂ is N, X₃ is N, A is

-   -    o is 0, and n is 0.    -   (13) In one embodiment, X is N, X₂ is N, X₃ is N, A is

-   -    is 0, n is 0, and t is 0.    -   (14) In one embodiment, X is N, X₂ is N, X₃ is N, A is

-   -    is 0, n is 0, t is 0, and r is 1.    -   (15) In one embodiment, X is N, X₂ is N, X₃ is N, r is 1, and R₄        is (C₁-C₄) haloalkyl.    -   (16) In one embodiment, X is N, X₂ is N, X₃ is N, r is 1, and R₄        is CF₃.

In one embodiment, the compound of Formula TL-I is of Formula IL-Ia orTL-Ib:

wherein X, X₂, X₃, R₁, R₂, R₃, R₄, n, o, t, and r are each as definedabove in Formula TL-I.

For a Targeting Ligand of Formula TL-Ia or TL-Ib:

-   -   (1) In one embodiment, X is N and X₂ is N.    -   (2) In one embodiment, X is N and X₃ is N.    -   (3) In one embodiment, X is N, X₂ is N, and X₃ is N.    -   (4) In one embodiment, X is N, X₂ is N, X₃ is N, and o is 0.    -   (5) In one embodiment, X is N, X₂ is N, X₃ is N, and n is 0.    -   (6) In one embodiment, X is N, X₂ is N, X₃ is N, and t is 0.    -   (7) In one embodiment, X is N, X₂ is N, X₃ is N, and r is 1.    -   (8) In one embodiment, X is N, X₂ is N, X₃ is N, o is 0, and n        is 0.    -   (9) In one embodiment, X is N, X₂ is N, X₃ is N, o is 0, n is 0,        and t is 0.    -   (10) In one embodiment, X is N, X₂ is N, X₃ is N, o is 0, n is        0, t is 0, and r is 1.    -   (11) In one embodiment, X is N, X₂ is N, X₃ is N, r is 1, and R₄        is (C₁-C₄) haloalkyl.    -   (12) In one embodiment, X is N, X₂ is N, X₃ is N, r is 1, and R₄        is CF₃.

X, X₂, X₃, R₁, R₂, R₃, R₄, n, o, t, and r can each be selected from anyof the groups and combined as described above in Formula TL-I.

In another embodiment, the compound of Formula TL-I is of Formula TL-Icor TL-Id:

wherein X, R₁, R₂, R₃, R₄, n, o, t, and r are each as defined above inFormula TL-I.

For a Targeting Ligand of Formula TL-Ic or TL-Id:

-   -   (1) In one embodiment, X is N and o is 0.    -   (2) In one embodiment, X is N and n is 0.    -   (3) In one embodiment, X is N and t is 0.    -   (4) In one embodiment, X is N and r is 1.    -   (5) In one embodiment, X is N, o is 0, and n is 0.    -   (6) In one embodiment, X is N, o is 0, n is 0, and t is 0.    -   (7) In one embodiment, X is N, o is 0, n is 0, t is 0, and r is        1.    -   (8) In one embodiment, X is N, r is 1, and R₄ is (C₁-C₄)        haloalkyl.    -   (9) In one embodiment, X is N, r is 1, and R₄ is CF₃.

X, R₂, R₃, R₄, n, o, t, and r can each be selected from any of thegroups and combined as described above in Formula TL-I.

In another embodiment, the compound of Formula TL-I is of Formula TL-Ieor IL-If:

wherein R₁, R₃, R₄, n, t, and r are each as defined above in FormulaTL-I.

For a Targeting Ligand of Formula TL-Ie or TL-If:

-   -   (1) In one embodiment, n is 0.    -   (2) In one embodiment, t is 0.    -   (3) In one embodiment, r is 1.    -   (4) In one embodiment, n is 0 and t is 0.    -   (5) In one embodiment, n is 0, t is 0, and r is 1.    -   (6) In one embodiment, r is 1 and R₄ is (C₁-C₄) haloalkyl.    -   (7) In one embodiment, r is 1 and R₄ is CF₃.

R₁, R₃, R₄, n, t, and r can each be selected from any of the groups andcombined as described above in Formula TL-I.

In one embodiment, a Targeting Ligand is a compound of Formula TL-II:

or an enantiomer, diastereomer, stereoisomer, or pharmaceuticallyacceptable salt thereof,wherein:

-   -   A′ is

-   -   B′ is

-   -   X₅ is N or CH;    -   X₆ is N or CH;    -   X₇ is N or CH;    -   R₅ is H or (C₁-C₄) alkyl;    -   each R₆ is independently (C₁-C₄) alkyl or (C₁-C₄) haloalkyl;    -   each R₇ is independently (C₁-C₄) alkyl, (C₁-C₄) haloalkyl,        (C₁-C₄) alkoxy, (C₁-C₄) haloalkoxy, or halogen;    -   each R₈ is independently (C₁-C₄) alkyl, (C₁-C₄) haloalkyl,        (C₁-C₄) alkoxy, (C₁-C₄) haloalkoxy, halogen, OH, or NH₂;    -   each R₉ is independently (C₁-C₄) alkyl, (C₁-C₄) haloalkyl,        (C₁-C₄) alkoxy, (C₁-C₄) haloalkoxy, halogen, OH, or NH₂;    -   n′ is 1 or 2;    -   r′ is 0, 1, or 2; and    -   o′, s, and s′ are each independently 0, 1, 2, or 3;        wherein the Targeting Ligand is bonded to the Linker via the

next to

In some embodiments, A′ is

In other embodiments, A′ is

In some embodiments, B′ is

In other embodiments, B′ is

In some embodiments, X₅ is N. In other embodiments, X₅ is CH.

In some embodiments, X₆ is N. In other embodiments, X₆ is CH.

In some embodiments, X₇ is N. In other embodiments, X₇ is CH.

In some embodiments, R₅ is H or (C₁-C₃) alkyl. In other embodiments, R₅is H or methyl, ethyl, propyl, or i-propyl. In other embodiments, R₅ isH, methyl, or ethyl. In other embodiments, R₅ is H or methyl. In otherembodiments, R₅ is H.

In some embodiments, each R₆ is independently (C₁-C₃) alkyl or (C₁-C₃)haloalkyl. In other embodiments, each R₆ is independently (C₁-C₃) alkyl.In other embodiments, each R₆ is independently (C₁-C₃) haloalkyl. Inother embodiments, each R₆ is independently (C₁-C₂) alkyl or (C₁-C₂)haloalkyl.

In some embodiments, each R₇ is independently (C₁-C₃) alkyl, (C₁-C₃)haloalkyl, (C₁-C₃) alkoxy, (C₁-C₃) haloalkoxy, or halogen. In otherembodiments, each R₇ is independently (C₁-C₃) alkyl or (C₁-C₃)haloalkyl. In other embodiments, each R₇ is independently (C₁-C₃) alkyl,(C₁-C₃) haloalkyl, or halogen. In other embodiments, each R₇ isindependently (C₁-C₃) alkyl. In other embodiments, each R₇ isindependently halogen. In other embodiments, each R₇ is independently(C₁-C₃) alkyl, (C₁-C₃) haloalkyl, (C₁-C₃) alkoxy, or (C₁-C₃) haloalkoxy.In other embodiments, each R₇ is independently (C₁-C₃) alkoxy, (C₁-C₃)haloalkoxy, or halogen.

In some embodiments, each R₈ is independently (C₁-C₃) alkyl, (C₁-C₃)haloalkyl, (C₁-C₃) alkoxy, (C₁-C₃) haloalkoxy, halogen, OH, or NH₂. Inother embodiments, each R₈ is independently halogen, OH, or NH₂. Inother embodiments, each R₈ is independently (C₁-C₃) alkyl, (C₁-C₃)haloalkyl, (C₁-C₃) alkoxy, (C₁-C₃) haloalkoxy, or halogen. In otherembodiments, each R₈ is independently (C₁-C₃) alkyl, (C₁-C₃) haloalkyl,(C₁-C₃) alkoxy, or (C₁-C₃) haloalkoxy. In other embodiments, each R₈ isindependently (C₁-C₃) alkyl or (C₁-C₃) haloalkyl. In other embodiments,each R₈ is independently (C₁-C₃) alkoxy or (C₁-C₃) haloalkoxy. In otherembodiments, each R₈ is independently halogen.

In some embodiments, each R₉ is independently (C₁-C₃) alkyl, (C₁-C₃)haloalkyl, (C₁-C₃) alkoxy, (C₁-C₃) haloalkoxy, halogen, OH, or NH₂. Inother embodiments, each R₉ is independently halogen, OH, or NH₂. Inother embodiments, each R₉ is independently (C₁-C₃) alkyl, (C₁-C₃)haloalkyl, (C₁-C₃) alkoxy, (C₁-C₃) haloalkoxy, or halogen. In otherembodiments, each R₉ is independently (C₁-C₃) alkyl, (C₁-C₃) haloalkyl,(C₁-C₃) alkoxy, or (C₁-C₃) haloalkoxy. In other embodiments, each R₉ isindependently (C₁-C₃) alkyl or (C₁-C₃) haloalkyl. In other embodiments,each R₉ is independently (C₁-C₃) alkoxy or (C₁-C₃) haloalkoxy. In otherembodiments, each R₉ is independently halogen. In other embodiments, atleast one R₉ is Cl.

In some embodiments, o′ is 0. In other embodiments, o′ is 1. In otherembodiments, o′ is 2. In other embodiments, o′ is 3. In otherembodiments, o′ is 0 or 1. In other embodiments, o′ is 1 or 2. In otherembodiments, o′ is 0, 1 or 2. In other embodiments, o′ is 1, 2, or 3.

In some embodiments, s is 0. In other embodiments, s is 1. In otherembodiments, s is 2. In other embodiments, s is 3. In other embodiments,s is 0 or 1. In other embodiments, s is 1 or 2. In other embodiments, sis 0, 1 or 2. In other embodiments, s is 1, 2, or 3.

In some embodiments, s′ is 0. In other embodiments, s′ is 1. In otherembodiments, s′ is 2. In other embodiments, s′ is 3. In otherembodiments, s′ is 0 or 1. In other embodiments, s′ is 1 or 2. In otherembodiments, s′ is 0, 1 or 2. In other embodiments, s′ is 1, 2, or 3.

In some embodiments, r′ is 0. In other embodiments, r′ is 1. In otherembodiments, r′ is 2. In other embodiments, r′ is 0 or 1. In otherembodiments, r′ is 1 or 2.

In other embodiments, n′ is 1. In other embodiments, n′ is 2.

Any of the groups described herein for any of A′, B′, X₅, X₆, X₇, R₅,R₆, R₇, R₈, R₉, n′, o′, r′, s, and s′ can be combined with any of thegroups described herein for one or more of the remainder of A′, B′, X₅,X₆, X₇, R₅, R₆, R₇, R₈, R₉, n′, o′, r′, s, and s′, and may further becombined with any of the groups described herein for the Linker.

For a Targeting Ligand of Formula TL-II:

-   -   (1) In one embodiment, X₅ is N and A′ is

-   -   (2) In one embodiment, X₅ is N and B′ is

-   -   (3) In one embodiment, X₅ is N, A′ is

and R₅ is H.

-   -   (4) In one embodiment, X₅ is N, B′ is

-   -    and R₅ is H.    -   (5) In one embodiment, X₅ is N, A′ is

-   -    and B′ is

-   -   (6) In one embodiment, X₅ is N, A′ is

-   -    B′ is

-   -    and R₅ is H.    -   (7) In one embodiment, X₅ is N and n′ is 1.    -   (8) In one embodiment, X₅ is N and o′ is 0.    -   (9) In one embodiment, X₅ is N and r′ is 0.    -   (10) In one embodiment, X₅ is N, n′ is 1, and o′ is 0.    -   (11) In one embodiment, X₅ is N, n′ is 1, and r′ is 0.    -   (12) In one embodiment, X₅ is N, n′ is 1, o′ is 0, and r′ is 0.    -   (13) In one embodiment, X₅ is N, n′ is 1, o′ is 0, r′ is 0, and        s is 0.    -   (14) In one embodiment, X₅ is N, A′ is

-   -    B′ is

-   -    R₅ is H, and R₉ is halogen.    -   (15) In one embodiment, X₅ is N, A′ is

-   -    B′ is

-   -    R₅ is H, R₉ is halogen, and n′ is 1.    -   (16) In one embodiment, X₅ is N, A′ is

-   -    B′ is

-   -    R₅ is H, R₉ is halogen, n′ is 1, and o′ is 0.    -   (17) In one embodiment, X₅ is N, A′ is

-   -    B′ is

-   -    R₅ is H, R₉ is halogen, n′ is 1, o′ is 0, and r′ is 0.    -   (18) In one embodiment, X₅ is N, A′ is

-   -    B′ is

-   -    R₅ is H, and n′ is 1.    -   (19) In one embodiment, X₅ is N, A′ is

-   -    B′ is

-   -    R₅ is H, n′ is 1, and o′ is 0.    -   (20) In one embodiment, X₅ is N, A′ is

-   -    B′ is

-   -    R₅ is H, n′ is 1, o′ is 0, and r′ is 0.

In one embodiment, the compound of Formula TL-II is of Formula TL-IIa orTL-IIb:

wherein A′, B′, X₅, R₅, R₆, R₇, n′, o′, and r′ are each as defined abovein Formula TL-II.

For a Targeting Ligand of Formula TL-IIa or TL-IIb:

-   -   (1) In one embodiment, X₅ is N and A′ is

-   -   (2) In one embodiment, X₅ is N and B′ is

-   -   (3) In one embodiment, X₅ is N, A′ is

-   -    and R₅ is H.    -   (4) In one embodiment, X₅ is N, B′ is

-   -    and R₅ is H.    -   (5)    -   (6) In one embodiment, X₅ is N, A′ is

-   -    and B′ is

-   -   (7) In one embodiment, X₅ is N, A′ is

-   -    B′ is

-   -    and R₅ is H.    -   (8) In one embodiment, X₅ is N and n′ is 1.    -   (9) In one embodiment, X₅ is N and o′ is 0.    -   (10) In one embodiment, X₅ is N and r′ is 0.    -   (11) In one embodiment, X₅ is N, n′ is 1, and o′ is 0.    -   (12) In one embodiment, X₅ is N, n′ is 1, and r′ is 0.    -   (13) In one embodiment, X₅ is N, n′ is 1, o′ is 0, and r′ is 0.    -   (14) In one embodiment, X₅ is N, n′ is 1, o′ is 0, r′ is 0, and        s is 0.    -   (15) In one embodiment, X₅ is N, A′ is

-   -    B′ is

-   -    R₅ is H, and R₉ is halogen.    -   (16) In one embodiment, X₅ is N, A′ is

-   -    B′ is

-   -    R₅ is H, R₉ is halogen, and n′ is 1.    -   (17) In one embodiment, X₅ is N, A′ is

-   -    B′ is

-   -    R₅ is H, R₉ is halogen, n′ is 1, and o′ is 0.    -   (18) In one embodiment, X₅ is N, A′ is

-   -    B′ is

-   -    R₅ is H, R₉ is halogen, n′ is 1, o′ is 0, and r′ is 0.    -   (19) In one embodiment, X₅ is N, A′ is

-   -    B′ is

-   -    R₅ is H, and n′ is 1.    -   (20) In one embodiment, X₅ is N, A′ is

-   -    B′ is

-   -    R₅ is H, n′ is 1, and o′ is 0.    -   (21) In one embodiment, X₅ is N, A′ is

-   -    B′ is

-   -    R₅ is H, n′ is 1, o′ is 0, and r′ is 0.

In one embodiment, the compound of Formula TL-II is of Formula TL-IIc orTL-IId:

wherein X₅, X₇, R₅, R₆, R₇, R₉, n′, o′, r′, and s′ are each as definedabove in Formula TL-II.

For a Targeting Ligand of Formula TL-IIc or TL-IId:

-   -   (1) In one embodiment, X₅ is N.    -   (2) In one embodiment, X₅ is N and R₅ is H.    -   (3) In one embodiment, X₅ is N and n′ is 1.    -   (4) In one embodiment, X₅ is N and o′ is 0.    -   (5) In one embodiment, X₅ is N and r′ is 0.    -   (6) In one embodiment, X₅ is N, n′ is 1, and o′ is 0.    -   (7) In one embodiment, X₅ is N, n′ is 1, and r′ is 0.    -   (8) In one embodiment, X₅ is N, n′ is 1, o′ is 0, and r′ is 0.    -   (9) In one embodiment, X₅ is N, n′ is 1, o′ is 0, r′ is 0, and s        is 0.    -   (10) In one embodiment, X₅ is N, R₅ is H, and R₉ is halogen.    -   (11) In one embodiment, X₅ is N, R₅ is H, R₉ is halogen, and n′        is 1.    -   (12) In one embodiment, X₅ is N, R₅ is H, R₉ is halogen, n′ is        1, and o′ is 0.    -   (13) In one embodiment, X₅ is N, R₅ is H, R₉ is halogen, n′ is        1, o′ is 0, and r′ is 0.    -   (14) In one embodiment, X₅ is N, R₅ is H, and n′ is 1.    -   (15) In one embodiment, X₅ is N, R₅ is H, n′ is 1, and o′ is 0.    -   (16) In one embodiment, X₅ is N, R₅ is H, n′ is 1, o′ is 0, and        r′ is 0.    -   (17) In one embodiment, X₅ is N, R₅ is H, n′ is 1, o′ is 0, r′        is 0, and s′ is 1.

In one embodiment, the compound of Formula TL-II is of Formula TL-IIc orTL-IId:

wherein X₅, R₅, R₇, R₉, n′, r′, and s′ are each as defined above inFormula TL-II.

For a Targeting Ligand of Formula TL-IIe or TL-IIf:

-   -   (1) In one embodiment, X₅ is N.    -   (2) In one embodiment, X₅ is N and R₅ is H.    -   (3) In one embodiment, X₅ is N and n′ is 1.    -   (4) In one embodiment, X₅ is N and s′ is 1.    -   (5) In one embodiment, X₅ is N and r′ is 0.    -   (6) In one embodiment, X₅ is N, n′ is 1, and s′ is 1.    -   (7) In one embodiment, X₅ is N, n′ is 1, and r′ is 0.    -   (8) In one embodiment, X₅ is N, n′ is 1, s′ is 1, and r′ is 0.    -   (9) In one embodiment, X₅ is N, R₅ is H, and R₉ is halogen.    -   (10) In one embodiment, X₅ is N, R₅ is H, R₉ is halogen, and n′        is 1.    -   (11) In one embodiment, X₅ is N, R₅ is H, R₉ is halogen, n′ is        1, and r′ is 0.    -   (12) In one embodiment, X₅ is N, R₅ is H, R₉ is halogen, n′ is        1, r′ is 0, and s′ is 1.    -   (13) In one embodiment, X₅ is N, R₅ is H, and n′ is 1.    -   (14) In one embodiment, X₅ is N, R₅ is H, n′ is 1, and r′ is 0.    -   (15) In one embodiment, X₅ is N, R₅ is H, n′ is 1, r′ is 0, and        s′ is 1.        Degron

The Degron serves to link a targeted protein, through a Linker and aTargeting Ligand, to a ubiquitin ligase for proteosomal degradation. Inone embodiment, the Degron is capable of binding to a ubiquitin ligase,such as an E3 ubiquitin ligase. In one embodiment, the Degron is capableof binding to cereblon.

In one embodiment, the Degron is of Formula D1:

or an enantiomer, diastereomer, or stereoisomer thereof, wherein:

-   -   Y is a bond, (CH₂)₁₋₆, (CH₂)₀₋₆—O, (CH₂)₀₋₆—C(O)NR₁₁,        (CH₂)₀₋₆—NR₁₁C(O), (CH₂)₀₋₆—NH, or (CH₂)₀₋₆—NR₁₂;    -   Z is C(O) or C(R₁₃)₂;    -   R₁₁ is H or C₁-C₆ alkyl;    -   R₁₂ is C₁-C₆ alkyl or C(O)—C₁-C₆ alkyl;    -   each R₁₃ is independently H or C₁-C₃ alkyl;    -   each R₁₄ is independently C₁-C₃ alkyl;    -   R₁₅ is H, deuterium, C₁-C₃ alkyl, F, or Cl;    -   each R₁₆ is independently halogen, OH, C₁-C₆ alkyl, or C₁-C₆        alkoxy;    -   q is 0, 1, or 2; and    -   v is 0, 1, 2, or 3,        wherein the Degron is covalently bonded to the Linker via

In one embodiment, Z is C(O).

In one embodiment, Z is C(R₁₃)₂; and each R₁₃ is H. In one embodiment, Xis C(R₁₃)₂; and one of R₁₃ is H, and the other is C₁-C₃ alkyl selectedfrom methyl, ethyl, and propyl. In one embodiment, Z is C(R₁₃)₂; andeach R₁₃ is independently selected from methyl, ethyl, and propyl.

In one embodiment, Y is a bond.

In one embodiment, Y is a bond, O, or NH.

In one embodiment, Y is (CH₂)₁, (CH₂)₂, (CH₂)₃, (CH₂)₄, (CH₂)₅, or(CH₂)₆. In one embodiment, Y is (CH₂)₁, (CH₂)₂, or (CH₂)₃. In oneembodiment, Y is (CH₂)₁ or (CH₂)₂.

In one embodiment, Y is O, CH₂—O, (CH₂)₂—O, (CH₂)₃—O, (CH₂)₄—O,(CH₂)₅—O, or (CH₂)₆—O. In one embodiment, Y is O, CH₂—O, (CH₂)₂—O, or(CH₂)₃—O. In one embodiment, Y is O or CH₂—O. In one embodiment, Y is O.

In one embodiment, Y is C(O)NR₁₁, CH₂—C(O)NR₁₁, (CH₂)₂—C(O)NR₁₁,(CH₂)₃—C(O)NR₁₁, (CH₂)₄—C(O)NR₁₁, (CH₂)₅—C(O)NR₁₁, or (CH₂)₆—C(O)NR₁₁.In one embodiment, Y is C(O)NR₁₁, CH₂—C(O)NR₁₁, (CH₂)₂—C(O)NR₁₁, or(CH₂)₃—C(O)NR₁₁. In one embodiment, Y is C(O)NR₁₁ or CH₂—C(O)NR₁₁. Inone embodiment, Y is C(O)NR₁₁.

In one embodiment, Y is NR₁₁C(O), CH₂—NR₁₁C(O), (CH₂)₂—NR₁₁C(O),(CH₂)₃—NR₁₁C(O), (CH₂)₄—NR₁₁C(O), (CH₂)₅—NR₁₁C(O), or (CH₂)₆—NR₁₁C(O).In one embodiment, Y is NR₁₁C(O), CH₂—NR₁₁C(O), (CH₂)₂—NR₁₁C(O), or(CH₂)₃—NR₁₁C(O). In one embodiment, Y is NR₁₁C(O) or CH₂—NR₁₁C(O). Inone embodiment, Y is NR₁₁C(O).

In one embodiment, R₁₁ is H. In one embodiment, R₁₁ is selected frommethyl, ethyl, propyl, butyl, i-butyl, t-butyl, pentyl, i-pentyl, andhexyl. In one embodiment, R₁₁ is C₁-C₃ alkyl selected from methyl,ethyl, and propyl.

In one embodiment, Y is NH, CH₂—NH, (CH₂)₂—NH, (CH₂)₃—NH, (CH₂)₄—NH,(CH₂)₅—NH, or (CH₂)₆—NH. In one embodiment, Y is NH, CH₂—NH, (CH₂)₂—NH,or (CH₂)₃—NH. In one embodiment, Y is NH or CH₂—NH. In one embodiment, Yis NH.

In one embodiment, Y is NR₁₂, CH₂—NR₁₂, (CH₂)₂—NR₁₂, (CH₂)₃—NR₁₂,(CH₂)₄—NR₁₂, (CH₂)₅—NR₁₂, or (CH₂)₆—NR₁₂. In one embodiment, Y is NR₁₂,CH₂—NR₁₂, (CH₂)₂—NR₁₂, or (CH₂)₃—NR₁₂. In one embodiment, Y is NR₁₂ orCH₂—NR₁₂. In one embodiment, Y is NR₁₂.

In one embodiment, R₁₂ is selected from methyl, ethyl, propyl, butyl,i-butyl, t-butyl, pentyl, i-pentyl, and hexyl. In one embodiment, R₁₂ isC₁-C₃ alkyl selected from methyl, ethyl, and propyl.

In one embodiment, R₁₂ is selected from C(O)-methyl, C(O)-ethyl,C(O)-propyl, C(O)-butyl, C(O)-i-butyl, C(O)-t-butyl, C(O)-pentyl,C(O)-i-pentyl, and C(O)-hexyl. In one embodiment, R₁₂ is C(O)—C₁-C₃alkyl selected from C(O)-methyl, C(O)-ethyl, and C(O)-propyl.

In one embodiment, R₁₃ is H.

In one embodiment, R₁₃ is C₁-C₃ alkyl selected from methyl, ethyl, andpropyl. In one embodiment, R₁₃ is methyl.

In one embodiment, q is 0.

In one embodiment, q is 1.

In one embodiment, q is 2.

In one embodiment, each R₁₄ is independently C₁-C₃ alkyl selected frommethyl, ethyl, and propyl.

In one embodiment, v is 0.

In one embodiment, v is 1.

In one embodiment, v is 2.

In one embodiment, v is 3.

In one embodiment, each R₁₆ is independently selected from halogen(e.g., F, Cl, Br, and I), OH, C₁-C₆ alkyl (e.g., methyl, ethyl, propyl,butyl, i-butyl, t-butyl, pentyl, i-pentyl, and hexyl), and C₁-C₆ alkoxy(e.g., methoxy, ethoxy, propoxy, butoxy, i-butoxy, t-butoxy, andpentoxy). In a further embodiment, each R₁₆ is independently selectedfrom F, Cl, OH, methyl, ethyl, propyl, butyl, i-butyl, t-butyl, methoxy,and ethoxy.

In one embodiment, R₁₅ is H, deuterium, or C₁-C₃ alkyl. In anotherembodiment, R₁₅ is H or C₁-C₃ alkyl. In a further embodiment, R₁₅ is inthe (S) or (R) configuration. In a further embodiment, R₁₅ is in the (S)configuration. In one embodiment, the compound comprises a racemicmixture of (S)—R₁₅ and (R)—R₁₅.

In one embodiment, R₁₅ is H.

In one embodiment, R₁₅ is deuterium.

In one embodiment, R₁₅ is C₁-C₃ alkyl selected from methyl, ethyl, andpropyl. In one embodiment, R₁₅ is methyl.

In one embodiment, R₁₅ is F or Cl. In a further embodiment, R₁₅ is inthe (S) or (R) configuration. In a further embodiment, R₁₅ is in the (R)configuration. In one embodiment, the compound comprises a racemicmixture of (S)—R₁₅ and (R)—R₁₅. In one embodiment, R₁₅ is F.

Any of the groups described herein for any of Y, Z, R₁₁, R₁₂, R₁₃, R₁₄,R₁₅, R₁₆, q and v can be combined with any of the groups describedherein for one or more of the remainder of Y, Z, R₁₁, R₁₂, R₁₃, R₁₄,R₁₅, R₁₆, q and v, and may further be combined with any of the groupsdescribed herein for the Linker.

For a Degron of Formula D1:

-   -   (1) In one embodiment, Z is C(O) and Y is a bond.    -   (2) In one embodiment, Z is C(O) and Y is NH.    -   (3) In one embodiment, Z is C(O) and Y is (CH₂)₀₋₆—O. In a        further embodiment, Y is O.    -   (4) In one embodiment, Z is C(O); Y is a bond; and q and v are        each 0.    -   (5) In one embodiment, Z is C(O); Y is NH; and q and v are each        0.    -   (6) In one embodiment, Z is C(O); Y is (CH₂)₀₋₆—O; and q and v        are each 0. In a further embodiment, Y is O.    -   (7) In one embodiment, Z is C(O); Y is a bond; and R₁₃ is H.    -   (8) In one embodiment, Z is C(O); Y is a bond; and R₁₅ is H.    -   (9) In one embodiment, Z is C(O); Y is NH; and R₁₃ is H.    -   (10) In one embodiment, Z is C(O); Y is NH; and R₁₅ is H.    -   (11) In one embodiment, Z is C(O); Y is a bond; R₁₃ is H; and        R₁₅ is H.    -   (12) In one embodiment, Z is C(O); Y is NH; R₁₃ is H; and R₁₅ is        H.    -   (13) In one embodiment, Z is C(O); Y is (CH₂)₀₋₆—O; and R₁₃        is H. In a further embodiment, Y is O.    -   (14) In one embodiment, Z is C(O); Y is (CH₂)₀₋₆—O; and R₁₅        is H. In a further embodiment, Y is O.    -   (15) In one embodiment, Z is C(O); Y is (CH₂)₀₋₆—O; R₁₃ is H;        and R₁₅ is H. In a further embodiment, Y is O.    -   (16) In one embodiment, q and v are each 0; and Y, Z, R₁₃, R₁₅,        and R₁₆ are each as defined in any of (1)-(3) and (7)-(15).

In one embodiment, the Degron is of Formula D1a or D1b:

or an enantiomer, diastereomer, or stereoisomer thereof, wherein Y, R₁₄,R₁₆, q, and v are each as defined above in Formula D1, and can beselected from any moieties or combinations thereof described above.

In one embodiment, Y is a bond, O, or NH. In one embodiment, Y is abond. In one embodiment, Y is O. In one embodiment, Y is NH.

In one embodiment, the Degron is of Formula D2:

or an enantiomer, diastereomer, or stereoisomer thereof, wherein:

-   -   each R₁₇ is independently C₁-C₃ alkyl;    -   q′ is 0, 1, 2, 3 or 4; and    -   R₁₈ is H or C₁-C₃ alkyl,        wherein the Degron is covalently bonded to another moiety (e.g.,        a compound, or a Linker) via

In one embodiment, q′ is 0.

In one embodiment, q′ is 1.

In one embodiment, q′ is 2.

In one embodiment, q′ is 3.

In one embodiment, each R₁₇ is independently C₁-C₃ alkyl selected frommethyl, ethyl, and propyl.

In one embodiment, Rig is methyl, ethyl, or propyl. In one embodiment,R₁₈ is methyl.

In one embodiment, the Degron is of Formula D2a or D2b:

Linker

The Linker is a bond or a carbon chain that serves to link a TargetingLigand with a Degron. In one embodiment, the carbon chain optionallycomprises one, two, three, or more heteroatoms selected from N, O, andS. In one embodiment, the carbon chain comprises only saturated chaincarbon atoms. In one embodiment, the carbon chain optionally comprisestwo or more unsaturated chain carbon atoms (e.g., C═C or C≡C). In oneembodiment, one or more chain carbon atoms in the carbon chain areoptionally substituted with one or more substituents (e.g., oxo, C₁-C₆alkyl, C₂-C₆ alkenyl, C₂-C₆ alkynyl, C₁-C₃ alkoxy, OH, halogen, NH₂,NH(C₁-C₃ alkyl), N(C₁-C₃ alkyl)₂, CN, C₃-C₈ cycloalkyl, heterocyclyl,phenyl, and heteroaryl).

In one embodiment, the Linker comprises at least 5 chain atoms (e.g., C,O, N, and S). In one embodiment, the Linker comprises less than 25 chainatoms (e.g., C, O, N, and S). In one embodiment, the Linker comprisesless than 20 chain atoms (e.g., C, O, N, and S). In one embodiment, theLinker comprises 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18,19, 20, 21, 22, 23, or 24 chain atoms (e.g., C, O, N, and S). In oneembodiment, the Linker comprises 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15,16, 17, 18, 19, 20, 21, 22, 23, or 24 chain atoms (e.g., C, O, N, andS). In one embodiment, the Linker comprises 5, 7, 9, 11, 13, 15, 17, or19 chain atoms (e.g., C, O, N, and S). In one embodiment, the Linkercomprises 5, 7, 9, or 11 chain atoms (e.g., C, O, N, and S). In oneembodiment, the Linker comprises 11, 13, 15, 17, or 19 chain atoms(e.g., C, O, N, and S). In one embodiment, the Linker comprises 11, 13,15, 17, 19, 21, or 23 chain atoms (e.g., C, O, N, and S). In oneembodiment, the Linker comprises 6, 8, 10, 12, 14, 16, 18, 20, 22, or 24chain atoms (e.g., C, O, N, and S). In one embodiment, the Linkercomprises 6, 8, 10, 12, 14, 16, 18, or 20 chain atoms (e.g., C, O, N,and S). In one embodiment, the Linker comprises 6, 8, 10, or 12 chainatoms (e.g., C, O, N, and S). In one embodiment, the Linker comprises12, 14, 16, 18, or 20 chain atoms (e.g., C, O, N, and S).

In one embodiment, the Linker comprises from 11 to 19 chain atoms (e.g.,C, O, N, and S).

In one embodiment, the Linker is a carbon chain optionally substitutedwith non-bulky substituents (e.g., oxo, C₁-C₆ alkyl, C₂-C₆ alkenyl,C₂-C₆ alkynyl, C₁-C₃ alkoxy, OH, halogen, NH₂, NH(C₁-C₃ alkyl), N(C₁-C₃alkyl)₂, and CN). In one embodiment, the non-bulky substitution islocated on the chain carbon atom proximal to the Degron (i.e., thecarbon atom is separated from the carbon atom to which the Degron isbonded by at least 3, 4, or 5 chain atoms in the Linker). In oneembodiment, the non-bulky substitution is located on the chain carbonatom proximal to the Targeting Ligand (i.e., the carbon atom isseparated from the carbon atom to which the Degron is bonded by at least3, 4, or 5 chain atoms in the Linker).

In one embodiment, the Linker is of Formula L0:

or an enantiomer, diastereomer, or stereoisomer thereof, wherein

-   -   p1 is an integer selected from 0 to 12;    -   p2 is an integer selected from 0 to 12;    -   p3 is an integer selected from 1 to 6;    -   each W is independently absent, CH₂, O, S, NH, or NR₁₉;    -   Z₁ is absent, C(O), (CH₂)_(j)C(O)NH, CH₂, O, NH, or NR₁₉;    -   each R₁₉ is independently C₁-C₃ alkyl;    -   j is 1, 2, or 3; and    -   Q is absent or NHC(O)CH₂,        wherein the Linker is covalently bonded to a Degron via

the next to Q, and covalently bonded to a Targeting Ligand via the

In one embodiment, the total number of chain atoms in the Linker is lessthan 30. In a further embodiment, the total number of chain atoms in theLinker is less than 20.

For a Linker of Formula L0:

In one embodiment, p1 is an integer selected from 0 to 10.

In one embodiment, p1 is an integer selected from 1 to 10.

In one embodiment, p1 is selected from 1, 2, 3, 4, 5, and 6.

In one embodiment, p1 is 0, 1, 3, or 5.

In one embodiment, p1 is 0, 1, 2, or 3.

In one embodiment, p1 is 0.

In one embodiment, p1 is 1.

In one embodiment, p1 is 3.

In one embodiment, p1 is 5.

In one embodiment, p2 is an integer selected from 0 to 10.

In one embodiment, p2 is selected from 0, 1, 2, 3, 4, 5, and 6.

In one embodiment, p2 is 0, 1, 2, or 3.

In one embodiment, p2 is 0.

In one embodiment, p2 is 1.

In one embodiment, p3 is an integer selected from 1 to 5.

In one embodiment, p3 is 2, 3, 4, or 5.

In one embodiment, p3 is 0, 1, 2, or 3.

In one embodiment, p3 is 0.

In one embodiment, p3 is 1.

In one embodiment, p3 is 2.

In one embodiment, at least one W is CH₂.

In one embodiment, at least one W is O.

In one embodiment, at least one W is S.

In one embodiment, at least one W is NH.

In one embodiment, at least one W is NR₁₉; and each R₁₉ is independentlyC₁-C₃ alkyl selected from methyl, ethyl, and propyl.

In one embodiment, each W is O.

In one embodiment, j is 1, 2, or 3.

In one embodiment, j is 1.

In one embodiment, j is 2.

In one embodiment, j is 3.

In one embodiment, j is 2 or 3.

In one embodiment, j is 1 or 2.

In one embodiment, Q is absent.

In one embodiment, Q is NHC(O)CH₂.

In one embodiment, Z₁ is absent.

In one embodiment, Z₁ is CH₂.

In one embodiment, Z₁ is O.

In one embodiment, Z₁ is C(O).

In one embodiment, Z₁ is (CH₂)_(j)C(O)NH.

In one embodiment, Z₁ is NR₁₉; and R₁₉ is C₁-C₃ alkyl selected frommethyl, ethyl, and propyl.

In one embodiment, Z₁ is part of the Targeting Ligand that is bonded tothe Linker, namely, Z₁ is formed from reacting a functional group of theTargeting Ligand with the Linker.

In one embodiment, p1 is 1, 2, 3, or 4. In one embodiment, p1 is 1. Inone embodiment, p1 is 2. In one embodiment, p1 is 3. In one embodiment,p1 is 4.

In one embodiment, p1 is 1 and Z₁ is absent.

In one embodiment, p1 is 1, Z₁ is absent, and W is CH₂.

In one embodiment, p1 is 1, Z₁ is absent, and p3 is 1.

In one embodiment, p1 is 1, Z₁ is absent, and p3 is 2.

In one embodiment, p1 is 1, Z₁ is absent, and p2 is 0.

In one embodiment, p1 is 1, Z₁ is absent, p3 is 2, and p2 is 0.

In one embodiment, p1 is 1, Z₁ is absent, p3 is 2, p2 is 0, and each Wis O.

In one embodiment, p1 is 1, Z₁ is absent, p3 is 2, p2 is 0, each W is O,and Q is absent.

In one embodiment, p1 is 3 and Z₁ is absent.

In one embodiment, p1 is 3, Z₁ is absent, and p3 is 2.

In one embodiment, p1 is 3, Z₁ is absent, and p2 is 0.

In one embodiment, p1 is 3, Z₁ is absent, p3 is 2, and p2 is 0.

In one embodiment, p1 is 3, Z₁ is absent, p3 is 2, p2 is 0, and each Wis O.

In one embodiment, p1 is 3, Z₁ is absent, p3 is 2, p2 is 0, each W is O,and Q is absent.

In one embodiment, p1 is 5 and Z₁ is absent.

In one embodiment, p1 is 5, Z₁ is absent, and p3 is 2.

In one embodiment, p1 is 5, Z₁ is absent, and p2 is 0.

In one embodiment, p1 is 5, Z₁ is absent, p3 is 2, and p2 is 0.

In one embodiment, p1 is 5, Z₁ is absent, p3 is 2, p2 is 0, and each Wis O.

In one embodiment, p1 is 5, Z₁ is absent, p3 is 2, p2 is 0, each W is O,and Q is absent.

In one embodiment, p1 is 1 and Z₁ is C(O).

In one embodiment, p1 is 1, Z₁ is C(O), and p3 is 2.

In one embodiment, p1 is 1, Z₁ is C(O), and p2 is 0.

In one embodiment, p1 is 1, Z₁ is C(O), p3 is 2, and p2 is 0.

In one embodiment, p1 is 1, Z₁ is C(O), p3 is 2, p2 is 0, and each W isO.

In one embodiment, p1 is 1, Z₁ is C(O), p3 is 2, p2 is 0, each W is O,and Q is absent.

In one embodiment, p1 is 3 and Z₁ is C(O).

In one embodiment, p1 is 3, Z₁ is C(O), and p3 is 2.

In one embodiment, p1 is 3, Z₁ is C(O), and p2 is 0.

In one embodiment, p1 is 3, Z₁ is C(O), p3 is 2, and p2 is 0.

In one embodiment, p1 is 3, Z₁ is C(O), p3 is 2, p2 is 0, and each W isO.

In one embodiment, p1 is 3, Z₁ is C(O), p3 is 2, p2 is 0, each W is O,and Q is absent.

In one embodiment, p1 is 5 and Z₁ is C(O).

In one embodiment, p1 is 5, Z₁ is C(O), and p3 is 2.

In one embodiment, p1 is 5, Z₁ is C(O), and p2 is 0.

In one embodiment, p1 is 5, Z₁ is C(O), p3 is 2, and p2 is 0.

In one embodiment, p1 is 5, Z₁ is C(O), p3 is 2, p2 is 0, and each W isO.

In one embodiment, p1 is 5, Z₁ is C(O), p3 is 2, p2 is 0, each W is O,and Q is absent.

In one embodiment, p3 is 3 and Z₁ is absent.

In one embodiment, p3 is 3, Z₁ is absent, and p1 is 0.

In one embodiment, p3 is 3, Z₁ is absent, p1 is 0, and Q is absent.

In one embodiment, p3 is 4 and Z₁ is absent.

In one embodiment, p3 is 4, Z₁ is absent, and p1 is 0.

In one embodiment, p3 is 4, Z₁ is absent, p1 is 0, and Q is absent.

In one embodiment, p3 is 2, and Z₁ is absent.

In one embodiment, p1 is 3 and Z₁ is (CH₂)_(j)C(O)NH.

In one embodiment, p1 is 3 and Z₁ is (CH₂)C(O)NH.

In one embodiment, p1 is 3 and Z₁ is (CH₂)₂C(O)NH.

In one embodiment, p1 is 3 and Z₁ is (CH₂)₃C(O)NH.

In one embodiment, p1 is 3, Z₁ is (CH₂)_(j)C(O)NH, and p3 is 2.

In one embodiment, p1 is 3, Z₁ is (CH₂)C(O)NH, and p3 is 2.

In one embodiment, p1 is 3, Z₁ is (CH₂)₂C(O)NH, and p3 is 2.

In one embodiment, p1 is 3, Z₁ is (CH₂)₃C(O)NH, and p3 is 2.

In one embodiment, p1 is 3, Z₁ is (CH₂)_(j)C(O)NH, p3 is 2, and p2 is 0.

In one embodiment, p1 is 3, Z₁ is (CH₂)C(O)NH, p3 is 2, and p2 is 0.

In one embodiment, p1 is 3, Z₁ is (CH₂)₂C(O)NH, p3 is 2, and p2 is 0.

In one embodiment, p1 is 3, Z₁ is (CH₂)₃C(O)NH, p3 is 2, and p2 is 0.

In one embodiment, p1 is 3, Z₁ is (CH₂)_(j)C(O)NH, p3 is 2, p2 is 0, andeach W is O.

In one embodiment, p1 is 3, Z₁ is (CH₂)C(O)NH, p3 is 2, p2 is 0, andeach W is O.

In one embodiment, p1 is 3, Z₁ is (CH₂)₂C(O)NH, p3 is 2, p2 is 0, andeach W is O.

In one embodiment, p1 is 3, Z₁ is (CH₂)₃C(O)NH, p3 is 2, p2 is 0, andeach W is O.

In one embodiment, p1 is 3, Z₁ is (CH₂)_(j)C(O)NH, p3 is 2, p2 is 0,each W is O, and Q is absent.

In one embodiment, p1 is 3, Z₁ is (CH₂)C(O)NH, p3 is 2, p2 is 0, each Wis O, and Q is absent.

In one embodiment, p1 is 3, Z₁ is (CH₂)₂C(O)NH, p3 is 2, p2 is 0, each Wis O, and Q is absent.

In one embodiment, p1 is 3, Z₁ is (CH₂)₃C(O)NH, p3 is 2, p2 is 0, each Wis O, and Q is absent.

In one embodiment, p1 is 3 and Z₁ is CH₂C(O)NH.

In one embodiment, p1 is 3, Z₁ is CH₂C(O)NH, and Q is absent.

In one embodiment, p1 is 4 and Z₁ is absent.

In one embodiment, p1 is 4, Z₁ is absent, and p2 is 1.

In one embodiment, p1 is 4, Z₁ is absent, p2 is 1, and Q is absent.

In one embodiment, p1 is 4, Z₁ is absent, p2 is 1, and p3 is 3.

In one embodiment, p1 is 4, Z₁ is absent, p2 is 1, p3 is 3, and Q isabsent.

In one embodiment, p1 is 3 and Z₁ is absent.

In one embodiment, p1 is 3, Z₁ is absent, and p3 is 3.

In one embodiment, p1 is 3, Q is absent, and p3 is 3.

In one embodiment, p1 is 4, Z₁ is absent, and p3 is 3.

In one embodiment, p1 is 4, Z₁ is absent, p3 is 3, and Q is absent.

In one embodiment, p1 is 4, Z₁ is absent, p3 is 3, Q is absent, and p2is 0.

In one embodiment, p1 is 4, Z₁ is absent, and Q is absent.

In one embodiment, p1 is 3, Z₁ is CH₂C(O)NH, and Q is absent.

In one embodiment, p1 is 3, Z₁ is CH₂C(O)NH, Q is absent, and p3 is 2.

In one embodiment, p1 is 4, Q is absent, and p3 is 1.

In one embodiment, p1 is 4, Q is absent, p3 is 1, and p2 is 0.

In one embodiment, p1 is 4, Q is absent, and p3 is 3.

In one embodiment, p1 is 4, Q is absent, p3 is 3, and p2 is 0.

In one embodiment, the Linker-Targeting Ligand (TL) has the structureselected from Table L:

TABLE L

(L1)

(L2)

(L3)

(L4)

(L5)

(L6)

(L7)wherein Q, TL, p1, p3, and j are each as described above.

Any one of the Degrons described herein can be covalently bound to anyone of the Linkers described herein. Any one of the Targeting Ligandsdescribed herein can be covalently bound to any one of the Linkersdescribed herein.

In one embodiment, the present application relates to the Degron-Linker(DL), wherein the Degron is of Formula D1, and the Linker is selectedfrom L1-L7. In one embodiment, the Degron is of Formula D1a or D1b, andthe Linker is selected from L1-L7. In one embodiment, the Degron is ofFormula D1a or D1b, and the Linker is L1, L2, or L3. In one embodiment,the Degron is of Formula D1a or D1b, and the Linker is L4, L5, L6, orL7. In one embodiment, the Degron is of Formula D1b, and the Linker isL1, L2, or L3. In one embodiment, the Degron is of Formula D1b, and theLinker is L4, L5, L6, or L7.

In one embodiment, the present application relates to the Degron-Linker(DL), wherein the Degron is of Formula D2, and the Linker is selectedfrom L1-L7. In one embodiment, the Degron is of Formula D2a or D2b, andthe Linker is selected from L1-L7. In one embodiment, the Degron is ofFormula D2a or D2b, and the Linker is L1, L2, or L3. In one embodiment,the Degron is of Formula D2a or D2b, and the Linker is L4, L5, L6, orL7.

In one embodiment, the Linker is designed and optimized based on SAR(structure-activity relationship) and X-ray crystallography of theTargeting Ligand with regard to the location of attachment for theLinker.

In one embodiment, the optimal Linker length and composition vary by theTargeting Ligand and can be estimated based upon X-ray structure of theTargeting Ligand bound to its target. Linker length and composition canbe also modified to modulate metabolic stability and pharmacokinetic(PK) and pharmacodynamics (PD) parameters.

Some embodiments of present application relate to the bifunctionalcompounds having the following structures in Table A:

TABLE A Cmpd No. Structure I-1

I-2

I-3

I-4

I-5

I-6

I-7

I-8

I-9

Some of the foregoing compounds can comprise one or more asymmetriccenters, and thus can exist in various isomeric forms, e.g.,stereoisomers and/or diastereomers. Accordingly, compounds of theapplication may be in the form of an individual enantiomer, diastereomeror geometric isomer, or may be in the form of a mixture ofstereoisomers. In one embodiment, the compounds of the application areenantiopure compounds. In another embodiment, mixtures of stereoisomersor diastereomers are provided.

Furthermore, certain compounds, as described herein, may have one ormore double bonds that can exist as either the Z or E isomer, unlessotherwise indicated. The application additionally encompasses thecompounds as individual Z/E isomers substantially free of other E/Zisomers and alternatively, as mixtures of various isomers.

In one embodiment, the present application relates to compounds thattarget proteins, such as CDK8 for degradation, which have numerousadvantages over inhibitors of protein function (e.g., kinase activity)and can a) overcome resistance in certain cases; b) prolong the kineticsof drug effect by destroying the protein, thus requiring resynthesis ofthe protein even after the compound has been metabolized; c) target allfunctions of a protein at once rather than a specific catalytic activityor binding event; d) expand the number of drug targets by including allproteins that a ligand can be developed for, rather than proteins whoseactivity (e.g., kinase activity) can be affected by a small moleculeinhibitor, antagonist or agonist; and e) have increased potency comparedto inhibitors due to the possibility of the small molecule actingcatalytically.

Some embodiments of the present application relate to degradation orloss of 30% to 100% of the target protein. Some embodiments relate tothe loss of 50-100% of the target protein. Other embodiments relate tothe loss of 75-95% of the targeted protein.

A bifunctional compound of the present application (e.g., a bifunctionalcompound of any of the formulae described herein, or selected from anybifunctional compounds described herein) is capable of modulating (e.g.,decreasing) the amount of a targeted protein (e.g., CDK8). Abifunctional compound of the present application (e.g., a bifunctionalcompound of any of the formulae described herein, or selected from anybifunctional compounds described herein) is also capable of degrading atargeted protein (e.g., CDK8) through the UPP pathway. Accordingly, abifunctional compound of the present application (e.g., a bifunctionalcompound of any of the formulae described herein, or selected from anybifunctional compounds described herein) is capable of treating orpreventing a disease or disorder in which CDK8 plays a role. Abifunctional compound of the present application (e.g., a bifunctionalcompound of any of the formulae described herein, or selected from anybifunctional compounds described herein) is also capable of treating orpreventing a disease or disorder in which CDK8 plays a role or in whichCDK8 is deregulated (e.g., overexpressed).

Modulation of CDK8 through UPP-mediated degradation by a bifunctionalcompound of the application, such as those described herein, provides anovel approach to the treatment, prevention, or amelioration of diseasesor disorders in which CDK8 plays a role including, but not limited to,cancer and metastasis, inflammation, arthritis, systemic lupuserythematosus, skin-related disorders, pulmonary disorders,cardiovascular disease, ischemia, neurodegenerative disorders, liverdisease, gastrointestinal disorders, viral and bacterial infections,central nervous system disorders, Alzheimer's disease, Parkinson'sdisease, Huntington's disease, amyotrophic lateral sclerosis, spinalcord injury, and peripheral neuropathy. Further, modulation of CDK8through UPP-mediated degradation by a bifunctional compound of theapplication, such as those described herein, also provides a newparadigm for treating, preventing, or ameliorating diseases or disordersin which CDK8 is deregulated.

In one embodiment, a bifunctional compound of the present application(e.g., a bifunctional compound of any of the formulae described herein,or selected from any bifunctional compounds described herein) is moreefficacious in treating a disease or condition (e.g., cancer) than, oris capable of treating a disease or condition resistant to, theTargeting Ligand, when the Targeting Ligand is administered alone (i.e.,not bonded to a Linker and a Degron). In one embodiment, a bifunctionalcompound of the present application (e.g., a bifunctional compound ofany of the formulae described herein, or selected from any bifunctionalcompounds described herein) is capable of modulating (e.g., decreasing)the amount of CDK8, and thus is useful in treating a disease orcondition (e.g., cancer) in which CDK8 plays a role.

In one embodiment, the bifunctional compound of the present applicationthat is more efficacious in treating a disease or condition than, or iscapable of treating a disease or condition resistant to, the TargetingLigand, when the Targeting Ligand is administered alone (i.e., notbonded to a Linker and a Degron), is more potent in inhibiting thegrowth of cells (e.g., cancer cells) or decreasing the viability ofcells (e.g., cancer cells), than the Targeting Ligand, when theTargeting Ligand is administered alone (i.e., not bonded to a Linker anda Degron). In one embodiment, the bifunctional compound inhibits thegrowth of cells (e.g., cancer cells) or decreases the viability of cells(e.g., cancer cells) at an IC₅₀ that is lower than the IC₅₀ of theTargeting Ligand (when the Targeting Ligand is administered alone (i.e.,not bonded to a Linker and a Degron)) for inhibiting the growth ordecreasing the viability of the cells. In one embodiment, the IC₅₀ ofthe bifunctional compound is at most 90%, 80%, 70%, 60%, 50%, 40%, 30%,20%, 10%, 8%, 5%, 4%, 3%, 2%, 1%, 0.8%, 0.5%, 0.4%, 0.3%, 0.2%, or 0.1%of the IC₅₀ of the Targeting Ligand. In one embodiment, the IC₅₀ of thebifunctional compound is at most 50%, 40%, 30%, 20%, 10%, 8%, 5%, 4%,3%, 2%, 1%, 0.8%, 0.5%, 0.4%, 0.3%, 0.2%, or 0.1% of the IC₅₀ of theTargeting Ligand. In one embodiment, the IC₅₀ of the bifunctionalcompound is at most 30%, 20%, 10%, 8%, 5%, 4%, 3%, 2%, 1%, 0.8%, 0.5%,0.4%, 0.3%, 0.2%, or 0.1% of the IC₅₀ of the Targeting Ligand. In oneembodiment, the IC₅₀ of the bifunctional compound is at most 10%, 8%,5%, 4%, 3%, 2%, 1%, 0.8%, 0.5%, 0.4%, 0.3%, 0.2%, or 0.1% of the IC₅₀ ofthe Targeting Ligand. In one embodiment, the IC₅₀ of the bifunctionalcompound is at most 5%, 4%, 3%, 2%, 1%, 0.8%, 0.5%, 0.4%, 0.3%, 0.2%, or0.1% of the IC₅₀ of the Targeting Ligand. In one embodiment, the IC₅₀ ofthe bifunctional compound is at most 2%, 1%, 0.8%, 0.5%, 0.4%, 0.3%,0.2%, or 0.1% of the IC₅₀ of the Targeting Ligand. In one embodiment,the IC₅₀ of the bifunctional compound is at most 1%, 0.8%, 0.5%, 0.4%,0.3%, 0.2%, or 0.1% of the IC₅₀ of the Targeting Ligand. In oneembodiment, the bifunctional compound inhibits the growth of cells(e.g., cancer cells) or decreases the viability of cells (e.g., cancercells) at an E_(max) that is lower than the E_(max) of the TargetingLigand (when the Targeting Ligand is administered alone (i.e., notbonded to a Linker and a Degron)) for inhibiting the growth ordecreasing the viability of the cells. In one embodiment, the E_(max) ofthe bifunctional compound is at most 90%, 80%, 70%, 60%, 50%, 40%, 30%,20%, 10%, 8%, 5%, 4%, 3%, 2%, or 1% of the E_(max) of the TargetingLigand. In one embodiment, the E_(max) of the bifunctional compound isat most 50%, 40%, 30%, 20%, 10%, 8%, 5%, 4%, 3%, 2%, or 1% of theE_(max) of the Targeting Ligand. In one embodiment, the E_(max) of thebifunctional compound is at most 90%, 80%, 70%, 60%, 50%, 40%, 30%, 20%,or 10% of the E_(max) of the Targeting Ligand.

In some embodiments, the inhibition of CDK8 activity is measured byIC₅₀.

In some embodiments, the inhibition of CDK8 activity is measured byEC₅₀.

Potency of the inhibitor can be determined by EC₅₀ value. A compoundwith a lower EC₅₀ value, as determined under substantially similarconditions, is a more potent inhibitor relative to a compound with ahigher EC₅₀ value. In some embodiments, the substantially similarconditions comprise determining a CDK8-dependent phosphorylation level,in vitro or in vivo (e.g., in cells expressing a wild-type CDK8, amutant CDK8, or a fragment of any thereof).

Potency of the inhibitor can also be determined by IC₅₀ value. Acompound with a lower IC₅₀ value, as determined under substantiallysimilar conditions, is a more potent inhibitor relative to a compoundwith a higher IC₅₀ value. In some embodiments, the substantially similarconditions comprise determining a CDK8-dependent phosphorylation level,in vitro or in vivo (e.g., in cells expressing a wild-type CDK8, amutant CDK8, or a fragment of any thereof).

In one embodiment, the bifunctional compounds of the present applicationare useful as anticancer agents, and thus may be useful in the treatmentof cancer, by effecting tumor cell death or inhibiting the growth oftumor cells. In certain exemplary embodiments, the disclosed anticanceragents are useful in the treatment of cancers and other proliferativedisorders, including, but not limited to breast cancer, cervical cancer,colon and rectal cancer, leukemia, lung cancer (e.g., non-small celllung cancer), melanoma, multiple myeloma, non-Hodgkin's lymphoma,ovarian cancer, pancreatic cancer, prostate cancer, gastric cancer,leukemias (e.g., myeloid, lymphocytic, myelocytic and lymphoblasticleukemias), malignant melanomas, and T-cell lymphoma.

A “selective CDK8 inhibitor,” can be identified, for example, bycomparing the ability of a compound to inhibit CDK8 kinase activity toits ability to inhibit the other members of the CDK kinase family orother kinases. For example, a substance may be assayed for its abilityto inhibit CDK8 kinase activity, as well as CDK1, CDK2, CDK4, CDK6,CDK7, CDK9, CDK11, CDK12, CDK13, CDK14 and other kinases. In someembodiments, the selectivity can be identified by measuring the EC₅₀ orIC₅₀ of the compounds.

In some embodiments, the bifunctional compounds of the presentapplication containing a Target Ligand inhibit CDK8 more selectivelyover other cyclin-dependent kinases and/or other kinases than the TargetLigand alone (i.e., a Target Ligand itself compared to the Target Ligandcovalently bound to a Linker and a Degron). In certain embodiments, thebifunctional compounds of the application are about 10%, about 20%,about 30%, about 40%, about 50%, about 60%, about 70%, about 80%, about90% or about 99% more selective at inhibiting CDK8 than the TargetLigand alone. In certain embodiments, the bifunctional compounds of theapplication are about 10%, about 20%, about 30%, about 40%, or about 50%more selective at inhibiting CDK8 than the Target Ligand alone. Incertain embodiments, the bifunctional compounds of the application areabout 20%, about 30%, about 40%, about 50% or about 60% more selectiveat inhibiting CDK8 than the Target Ligand alone. In certain embodiments,the bifunctional compounds of the application are about 30%, about 40%,about 50%, about 60% or about 70% more selective at inhibiting CDK8 thanthe Target Ligand alone. In certain embodiments, the bifunctionalcompounds of the application are about 40%, about 50%, about 60%, about70%, or about 80% more selective at inhibiting CDK8 than the TargetLigand alone. In certain embodiments, the bifunctional compounds of theapplication are about 50%, about 60%, about 70%, about 80%, or about 90%more selective at inhibiting CDK8 than the Target Ligand alone. Incertain embodiments, the bifunctional compounds of the application areabout 60%, about 70%, about 80%, about 90%, or about 99% more selectiveat inhibiting CDK8 than the Target Ligand alone. In other embodiments,the bifunctional compounds of the application are at least 10%, at least20%, at least 30%, at least 40%, at least 50%, at least 60%, at least70%, at least 80%, at least 90%, or at least 99% more selective atinhibiting CDK8 than the Target Ligand alone.

In other embodiments, the bifunctional compounds of the application arebetween about 10% and about 99% more selective at inhibiting CDK8 thanthe Target Ligand alone. In other embodiments, the bifunctionalcompounds of the application are between about 10% and about 30% moreselective at inhibiting CDK8 than the Target Ligand alone. In otherembodiments, the bifunctional compounds of the application are betweenabout 20% and about 40% more selective at inhibiting CDK8 than theTarget Ligand alone. In other embodiments, the bifunctional compounds ofthe application are between about 30% and about 50% more selective atinhibiting CDK8 than the Target Ligand alone. In other embodiments, thebifunctional compounds of the application are between about 40% andabout 60% more selective at inhibiting CDK8 than the Target Ligandalone. In other embodiments, the bifunctional compounds of theapplication are between about 50% and about 70% more selective atinhibiting CDK8 than the Target Ligand alone. In other embodiments, thebifunctional compounds of the application are between about 60% andabout 80% more selective at inhibiting CDK8 than the Target Ligandalone. In other embodiments, the bifunctional compounds of theapplication are between about 70% and about 90% more selective atinhibiting CDK8 than the Target Ligand alone. In other embodiments, thebifunctional compounds of the application are between about 80% andabout 99% more selective at inhibiting CDK8 than the Target Ligandalone.

In some embodiments, the compounds of the present application areselective over other kinases. As used herein, “selective”, “selectiveCDK8 inhibitor”, or “selective CDK8 compound” refers to a compound, forexample a bifunctional compound of the application, that effectivelyinhibits CDK8 kinase to a greater extent than any other kinase enzyme,particularly any enzyme from the Cyclic-dependent kinase family (e.g.,CDK1, CDK2, CDK4, CDK6, CDK7, CDK9, CDK11, CDK12, CDK13, CDK14, etc.).

In certain embodiments, the compounds of the application are CDK8inhibitors that exhibit at least 2-fold, 3-fold, 5-fold, 10-fold,25-fold, 50-fold or 100-fold selectivity over other kinases (e.g., CDK1,CDK2, CDK4, CDK6, CDK7, CDK9, CDK11, CDK12, CDK13, CDK14, etc.). Invarious embodiments, the compounds of the application exhibit 1000-foldselectivity over other kinases.

In certain embodiments, the compounds of the application are CDK8inhibitors that exhibit at least 2-fold, 3-fold, 5-fold, 10-fold,25-fold, 50-fold or 100-fold selectivity over other cyclin-dependentkinases (e.g., CDK1, CDK2, CDK4, CDK6, CDK7, CDK9, CDK11, CDK12, CDK13,CDK14, etc.). In various embodiments, the compounds of the applicationexhibit 1000-fold selectivity over other cyclin-dependent kinases.

Definitions

Listed below are definitions of various terms used in this application.These definitions apply to the terms as they are used throughout thisspecification and claims, unless otherwise limited in specificinstances, either individually or as part of a larger group.

The term “alkyl,” as used herein, refers to saturated, straight orbranched-chain hydrocarbon radicals containing, in certain embodiments,between one and six carbon atoms. Examples of C₁-C₆ alkyl radicalsinclude, but are not limited to, methyl, ethyl, propyl, isopropyl,n-butyl, tert-butyl, neopentyl, and n-hexyl radicals.

The term “alkenyl,” as used herein, denotes a monovalent group derivedfrom a hydrocarbon moiety containing, in certain embodiments, from twoto six carbon atoms having at least one carbon-carbon double bond. Thedouble bond may or may not be the point of attachment to another group.Alkenyl groups include, but are not limited to, for example, ethenyl,propenyl, butenyl, 1-methyl-2-buten-1-yl and the like.

The term “alkoxy” refers to an —O-alkyl radical.

The terms “hal,” “halo,” and “halogen,” as used herein, refer to an atomselected from fluorine, chlorine, bromine and iodine.

The term “aryl,” as used herein, refers to a mono- or poly-cycliccarbocyclic ring system having one or more aromatic rings, fused ornon-fused, including, but not limited to, phenyl, naphthyl,tetrahydronaphthyl, indanyl, indenyl and the like.

The term “aralkyl,” as used herein, refers to an alkyl residue attachedto an aryl ring. Examples include, but are not limited to, benzyl,phenethyl and the like.

The term “cycloalkyl,” as used herein, denotes a monovalent groupderived from a monocyclic or polycyclic saturated or partiallyunsaturated carbocyclic ring compound. Examples of C₃-C₈ cycloalkylinclude, but not limited to, cyclopropyl, cyclobutyl, cyclopentyl,cyclohexyl, cyclopentyl and cyclooctyl; and examples ofC₃-C₁₂-cycloalkyl include, but not limited to, cyclopropyl, cyclobutyl,cyclopentyl, cyclohexyl, bicyclo [2.2.1] heptyl, and bicyclo [2.2.2]octyl. Also contemplated is a monovalent group derived from a monocyclicor polycyclic carbocyclic ring compound having at least onecarbon-carbon double bond by the removal of a single hydrogen atom.Examples of such groups include, but are not limited to, cyclopropenyl,cyclobutenyl, cyclopentenyl, cyclohexenyl, cycloheptenyl, cyclooctenyl,and the like.

The term “heteroaryl,” as used herein, refers to a mono- or poly-cyclic(e.g., bi-, or tri-cyclic or more) fused or non-fused, radical or ringsystem having at least one aromatic ring, having from five to ten ringatoms of which one ring atoms is selected from S, O, and N; zero, one,or two ring atoms are additional heteroatoms independently selected fromS, O, and N; and the remaining ring atoms are carbon. Heteroarylincludes, but is not limited to, pyridinyl, pyrazinyl, pyrimidinyl,pyrrolyl, pyrazolyl, imidazolyl, thiazolyl, oxazolyl, isooxazolyl,thiadiazolyl, oxadiazolyl, thiophenyl, furanyl, quinolinyl,isoquinolinyl, benzimidazolyl, benzooxazolyl, quinoxalinyl, and thelike.

The term “heteroaralkyl,” as used herein, refers to an alkyl residueattached to a heteroaryl ring. Examples include, but are not limited to,pyridinylmethyl, pyrimidinylethyl and the like.

The term “heterocyclyl,” or “heterocycloalkyl,” as used herein, refersto a non-aromatic 3-, 4-, 5-, 6- or 7-membered ring or a bi- ortri-cyclic group fused of non-fused system, where (i) each ring containsbetween one and three heteroatoms independently selected from oxygen,sulfur and nitrogen, (ii) each 5-membered ring has 0 to 1 double bondsand each 6-membered ring has 0 to 2 double bonds, (iii) the nitrogen andsulfur heteroatoms may optionally be oxidized, and (iv) the nitrogenheteroatom may optionally be quaternized. Representativeheterocycloalkyl groups include, but are not limited to, [1,3]dioxolane,pyrrolidinyl, pyrazolinyl, pyrazolidinyl, imidazolinyl, imidazolidinyl,piperidinyl, piperazinyl, oxazolidinyl, isoxazolidinyl, morpholinyl,thiazolidinyl, isothiazolidinyl, and tetrahydrofuryl.

The term “alkylamino” refers to a group having the structure —NH(C₁-C₁₂alkyl), e.g., —NH(C₁-C₆ alkyl), where C₁-C₁₂ alkyl is as previouslydefined.

The term “dialkylamino” refers to a group having the structure —N(C₁-C₁₂alkyl)₂, e.g., —NH(C₁-C₆ alkyl), where C₁-C₁₂ alkyl is as previouslydefined.

The term “acyl” includes residues derived from acids, including but notlimited to carboxylic acids, carbamic acids, carbonic acids, sulfonicacids, and phosphorous acids. Examples include aliphatic carbonyls,aromatic carbonyls, aliphatic sulfonyls, aromatic sulfinyls, aliphaticsulfinyls, aromatic phosphates and aliphatic phosphates. Examples ofaliphatic carbonyls include, but are not limited to, acetyl, propionyl,2-fluoroacetyl, butyryl, 2-hydroxy acetyl, and the like.

In accordance with the application, any of the aryls, substituted aryls,heteroaryls and substituted heteroaryls described herein, can be anyaromatic group. Aromatic groups can be substituted or unsubstituted.

The terms “hal,” “halo,” and “halogen,” as used herein, refer to an atomselected from fluorine, chlorine, bromine and iodine.

As described herein, compounds of the application may optionally besubstituted with one or more substituents, such as are illustratedgenerally above, or as exemplified by particular classes, subclasses,and species of the application. It will be appreciated that the phrase“optionally substituted” is used interchangeably with the phrase“substituted or unsubstituted.” In general, the term “substituted”,whether preceded by the term “optionally” or not, refers to thereplacement of hydrogen radicals in a given structure with the radicalof a specified substituent. Unless otherwise indicated, an optionallysubstituted group may have a substituent at each substitutable positionof the group, and when more than one position in any given structure maybe substituted with more than one substituent selected from a specifiedgroup, the substituent may be either the same or different at everyposition. The terms “optionally substituted”, “optionally substitutedalkyl,” “optionally substituted “optionally substituted alkenyl,”“optionally substituted alkynyl”, “optionally substituted cycloalkyl,”“optionally substituted cycloalkenyl,” “optionally substituted aryl”,“optionally substituted heteroaryl,” “optionally substituted aralkyl”,“optionally substituted heteroaralkyl,” “optionally substitutedheterocycloalkyl,” and any other optionally substituted group as usedherein, refer to groups that are substituted or unsubstituted byindependent replacement of one, two, or three or more of the hydrogenatoms thereon with substituents including, but not limited to:

-   -   —F, —CI, —Br, —I, —OH, protected hydroxy, —NO₂, —CN, —NH₂,        protected amino, —NH—C₁-C₁₂-alkyl, —NH—C₂-C₁₂-alkenyl,        —NH—C₂-C₁₂-alkenyl, —NH—C₃-C₁₂-cycloalkyl, —NH-aryl,        —NH-heteroaryl, —NH-heterocycloalkyl, -dialkylamino,        -diarylamino, -diheteroarylamino, —O—C₁-C₁₂-alkyl,        —O—C₂-C₁₂-alkenyl, —O—C₂-C₁₂-alkenyl, —O—C₃-C₁₂-cycloalkyl,        —O-aryl, —O-heteroaryl, —O-heterocycloalkyl, —C(O)—C₁-C₁₂-alkyl,        —C(O)—C₂-C₁₂-alkenyl, —C(O)—C₂-C₁₂-alkenyl,        —C(O)—C₃-C₁₂-cycloalkyl, —C(O)-aryl, —C(O)— heteroaryl,        —C(O)-heterocycloalkyl, —CONH₂, —CONH—C₁-C₁₂-alkyl,        —CONH—C₂-C₁₂-alkenyl, —CONH—C₂-C₁₂-alkenyl,        —CONH—C₃-C₁₂-cycloalkyl, —CONH-aryl, —CONH-heteroaryl,        —CONH-heterocycloalkyl, —OCO₂—C₁-C₁₂-alkyl,        —OCO₂—C₂-C₁₂-alkenyl, —OCO₂—C₂-C₁₂-alkenyl,        —OCO₂—C₃-C₁₂-cycloalkyl, —OCO₂-aryl, —OCO₂-heteroaryl,        —OCO₂-heterocycloalkyl, —OCONH₂, —OCONH—C₁-C₁₂-alkyl,        —OCONH—C₂-C₁₂-alkenyl, —OCONH—C₂-C₁₂-alkenyl,        —OCONH—C₃-C₁₂-cycloalkyl, —OCONH-aryl, —OCONH-heteroaryl,        —OCONH— heterocycloalkyl, —NHC(O)—C₁-C₁₂-alkyl,        —NHC(O)—C₂-C₁₂-alkenyl, —NHC(O)—C₂-C₁₂-alkenyl,        —NHC(O)—C₃-C₁₂-cycloalkyl, —NHC(O)-aryl, —NHC(O)-heteroaryl,        —NHC(O)— heterocycloalkyl, —NHCO₂—C₁-C₁₂-alkyl,        —NHCO₂—C₂-C₁₂-alkenyl, —NHCO₂—C₂-C₁₂-alkenyl,        —NHCO₂—C₃-C₁₂-cycloalkyl, —NHCO₂-aryl, —NHCO₂-heteroaryl,        —NHCO₂— heterocycloalkyl, NHC(O)NH₂, —NHC(O)NH—C₁-C₁₂-alkyl,        —NHC(O)NH—C₂-C₁₂-alkenyl, —NHC(O)NH—C₂-C₁₂-alkenyl,        —NHC(O)NH—C₃-C₁₂-cycloalkyl, —NHC(O)NH-aryl,        —NHC(O)NH-heteroaryl, NHC(O)NH-heterocycloalkyl, —NHC(S)NH₂,        —NHC(S)NH—C₁-C₁₂-alkyl, —NHC(S)NH—C₂-C₁₂-alkenyl,        —NHC(S)NH—C₂-C₁₂-alkenyl, —NHC(S)NH—C₃-C₁₂-cycloalkyl,        —NHC(S)NH-aryl, —NHC(S)NH-heteroaryl,        —NHC(S)NH-heterocycloalkyl, —NHC(NH)NH₂,        —NHC(NH)NH—C₁-C₁₂-alkyl, —NHC(NH)NH—C₂-C₁₂-alkenyl,        —NHC(NH)NH—C₂-C₁₂-alkenyl, —NHC(NH)NH—C₃-C₁₂-cycloalkyl,        —NHC(NH)NH-aryl, —NHC(NH)NH-heteroaryl,        —NHC(NH)NHheterocycloalkyl, —NHC(NH)—C₁-C₁₂-alkyl,        —NHC(NH)—C₂-C₁₂-alkenyl, —NHC(NH)—C₂-C₁₂-alkenyl,        —NHC(NH)—C₃-C₁₂-cycloalkyl, —NHC(NH)-aryl, —NHC(NH)-heteroaryl,        —NHC(NH)-heterocycloalkyl, —C(NH)NH—C₁-C₁₂-alkyl,        —C(NH)NH—C₂-C₁₂-alkenyl, —C(NH)NH—C₂-C₁₂-alkenyl,        C(NH)NH—C₃-C₁₂-cycloalkyl, —C(NH)NH-aryl, —C(NH)NH-heteroaryl,        —C(NH)NHheterocycloalkyl, —S(O)—C₁-C₁₂-alkyl,        —S(O)—C₂-C₁₂-alkenyl, —S(O)—C₂-C₁₂-alkenyl,        —S(O)—C₃-C₁₂-cycloalkyl, —S(O)-aryl, —S(O)-heteroaryl,        —S(O)-heterocycloalkyl —SO₂NH₂, —SO₂NH—C₁-C₁₂-alkyl,        —SO₂NH—C₂-C₁₂-alkenyl, —SO₂NH—C₂-C₁₂-alkenyl,        —SO₂NH—C₃-C₁₂-cycloalkyl, —SO₂NH-aryl, —SO₂NH-heteroaryl,        —SO₂NH-heterocycloalkyl, —NHSO₂—C₁-C₁₂-alkyl,        —NHSO₂—C₂-C₁₂-alkenyl, —NHSO₂—C₂-C₁₂-alkenyl,        —NHSO₂—C₃-C₁₂-cycloalkyl, —NHSO₂-aryl, —NHSO₂-heteroaryl,        —NHSO₂-heterocycloalkyl, —CH₂NH₂, —CH₂SO₂CH₃, -aryl, -arylalkyl,        -heteroaryl, -heteroarylalkyl, -heterocycloalkyl,        —C₃-C₁₂-cycloalkyl, polyalkoxyalkyl, polyalkoxy,        -methoxymethoxy, -methoxyethoxy, —SH, —S—C₁-C₁₂-alkyl,        —S—C₂-C₁₂-alkenyl, —S—C₂-C₁₂-alkenyl, —S—C₃-C₁₂-cycloalkyl,        —S-aryl, —S-heteroaryl, —S-heterocycloalkyl, or        methylthiomethyl.

It is understood that the aryls, heteroaryls, alkyls, and the like canbe substituted. The term “cancer” includes, but is not limited to, thefollowing cancers: epidermoid Oral: buccal cavity, lip, tongue, mouth,pharynx; Cardiac: sarcoma (angiosarcoma, fibrosarcoma, rhabdomyosarcoma,liposarcoma), myxoma, rhabdomyoma, fibroma, lipoma, and teratoma; Lung:bronchogenic carcinoma (squamous cell or epidermoid, undifferentiatedsmall cell, undifferentiated large cell, adenocarcinoma), alveolar(bronchiolar) carcinoma, bronchial adenoma, sarcoma, lymphoma,chondromatous hamartoma, mesothelioma; Gastrointestinal: esophagus(squamous cell carcinoma, larynx, adenocarcinoma, leiomyosarcoma,lymphoma), stomach (carcinoma, lymphoma, leiomyosarcoma), pancreas(ductal adenocarcinoma, insulinoma, glucagonoma, gastrinoma, carcinoidtumors, vipoma), small bowel or small intestines (adenocarcinoma,lymphoma, carcinoid tumors, Karposi's sarcoma, leiomyoma, hemangioma,lipoma, neurofibroma, fibroma), large bowel or large intestines(adenocarcinoma, tubular adenoma, villous adenoma, hamartoma,leiomyoma), colon, colonrectum, colorectal, rectum; Genitourinary tract:kidney (adenocarcinoma, Wilm's tumor (nephroblastoma), lymphoma,leukemia), bladder and urethra (squamous cell carcinoma, transitionalcell carcinoma, adenocarcinoma), prostate (adenocarcinoma, sarcoma),testis (seminoma, teratoma, embryonal carcinoma, teratocarcinoma,choriocarcinoma, sarcoma, interstitial cell carcinoma, fibroma,fibroadenoma, adenomatoid tumors, lipoma); Liver: hepatoma(hepatocellular carcinoma), cholangiocarcinoma, hepatoblastoma,angiosarcoma, hepatocellular adenoma, hemangioma, biliary passages;Bone: osteogenic sarcoma (osteosarcoma), fibrosarcoma, malignant fibroushistiocytoma, chondrosarcoma, Ewing's sarcoma, malignant lymphoma(reticulum cell sarcoma), multiple myeloma, malignant giant cell tumorchordoma, osteochronfroma (osteocartilaginous exostoses), benignchondroma, chondroblastoma, chondromyxofibroma, osteoid osteoma andgiant cell tumors; Nervous system: skull (osteoma, hemangioma,granuloma, xanthoma, osteitis deformans), meninges (meningioma,meningiosarcoma, gliomatosis), brain (astrocytoma, medulloblastoma,glioma, ependymoma, germinoma (pinealoma), glioblastoma multiform,oligodendroglioma, schwannoma, retinoblastoma, congenital tumors),spinal cord neurofibroma, meningioma, glioma, sarcoma); Gynecological:uterus (endometrial carcinoma), cervix (cervical carcinoma, pre-tumorcervical dysplasia), ovaries (ovarian carcinoma (serouscystadenocarcinoma, mucinous cystadenocarcinoma, unclassifiedcarcinoma), granulosa-thecal cell tumors, Sertoli-Leydig cell tumors,dysgerminoma, malignant teratoma), vulva (squamous cell carcinoma,intraepithelial carcinoma, adenocarcinoma, fibrosarcoma, melanoma),vagina (clear cell carcinoma, squamous cell carcinoma, botryoid sarcoma(embryonal rhabdomyosarcoma), fallopian tubes (carcinoma), breast;Hematologic: blood (myeloid leukemia (acute and chronic), acutelymphoblastic leukemia, chronic lymphocytic leukemia, myeloproliferativediseases, multiple myeloma, myelodysplastic syndrome), Hodgkin'sdisease, non-Hodgkin's lymphoma (malignant lymphoma) hairy cell;lymphoid disorders; Skin: malignant melanoma, basal cell carcinoma,squamous cell carcinoma, Karposi's sarcoma, keratoacanthoma, molesdysplastic nevi, lipoma, angioma, dermatofibroma, keloids, psoriasis,Thyroid gland: papillary thyroid carcinoma, follicular thyroidcarcinoma; medullary thyroid carcinoma, undifferentiated thyroid cancer,multiple endocrine neoplasia type 2A, multiple endocrine neoplasia type2B, familial medullary thyroid cancer, pheochromocytoma, paraganglioma;and Adrenal glands: neuroblastoma. Thus, the term “cancerous cell” asprovided herein, includes a cell afflicted by any one of theabove-identified conditions.

The term “CDK8” herein refers to cyclin-dependent kinase 8.

The term “subject” as used herein refers to a mammal. A subjecttherefore refers to, for example, dogs, cats, horses, cows, pigs, guineapigs, and the like. Preferably the subject is a human. When the subjectis a human, the subject may be referred to herein as a patient.

“Treat”, “treating” and “treatment” refer to a method of alleviating orabating a disease and/or its attendant symptoms.

As used herein, “preventing” or “prevent” describes reducing oreliminating the onset of the symptoms or complications of the disease,condition or disorder.

The term “targeted protein(s)” is used interchangeably with “targetprotein(s)”, unless the context clearly dictates otherwise. In oneembodiment, a “targeted protein” is CDK.

The term “subject” as used herein refers to a mammal. A subjecttherefore refers to, for example, dogs, cats, horses, cows, pigs, guineapigs, and the like. Preferably the subject is a human. When the subjectis a human, the subject may be referred to herein as a patient.

The terms “disease(s)”, “disorder(s)”, and “condition(s)” are usedinterchangeably, unless the context clearly dictates otherwise.

The term “therapeutically effective amount” of a bifunctional compoundor pharmaceutical composition of the application, as used herein, meansa sufficient amount of the bifunctional compound or pharmaceuticalcomposition so as to decrease the symptoms of a disorder in a subject.As is well understood in the medical arts a therapeutically effectiveamount of a bifunctional compound or pharmaceutical composition of thisapplication will be at a reasonable benefit/risk ratio applicable to anymedical treatment. It will be understood, however, that the total dailyusage of the compounds and compositions of the present application willbe decided by the attending physician within the scope of sound medicaljudgment. The specific inhibitory dose for any particular patient willdepend upon a variety of factors including the disorder being treatedand the severity of the disorder; the activity of the specific compoundemployed; the specific composition employed; the age, body weight,general health, sex and diet of the patient; the time of administration,route of administration, and rate of excretion of the specific compoundemployed; the duration of the treatment; drugs used in combination orcoincidental with the specific compound employed; and like factors wellknown in the medical arts.

As used herein, the term “pharmaceutically acceptable salt” refers tothose salts of the compounds formed by the process of the presentapplication which are, within the scope of sound medical judgment,suitable for use in contact with the tissues of humans and lower animalswithout undue toxicity, irritation, allergic response and the like, andare commensurate with a reasonable benefit/risk ratio. Pharmaceuticallyacceptable salts are well known in the art. For example, S. M. Berge, etal. describes pharmaceutically acceptable salts in detail in J.Pharmaceutical Sciences, 66: 1-19 (1977). The salts can be prepared insitu during the final isolation and purification of the compounds of theapplication, or separately by reacting the free base or acid functionwith a suitable acid or base.

Examples of pharmaceutically acceptable salts include, but are notlimited to, nontoxic acid addition salts: salts formed with inorganicacids such as hydrochloric acid, hydrobromic acid, phosphoric acid,sulfuric acid and perchloric acid, or with organic acids such as aceticacid, maleic acid, tartaric acid, citric acid, succinic acid or malonicacid. Other pharmaceutically acceptable salts include, but are notlimited to, adipate, alginate, ascorbate, aspartate, benzenesulfonate,benzoate, bisulfate, borate, butyrate, camphorate, camphorsulfonate,citrate, cyclopentanepropionate, digluconate, dodecylsulfate,ethanesulfonate, formate, fumarate, glucoheptonate, glycerophosphate,gluconate, hemisulfate, heptanoate, hexanoate, hydroiodide,2-hydroxy-ethanesulfonate, lactobionate, lactate, laurate, laurylsulfate, malate, maleate, malonate, methanesulfonate,2-naphthalenesulfonate, nicotinate, nitrate, oleate, oxalate, palmitate,pamoate, pectinate, persulfate, 3-phenylpropionate, phosphate, picrate,pivalate, propionate, stearate, succinate, sulfate, tartrate,thiocyanate, /7-toluenesulfonate, undecanoate, valerate salts, and thelike. Representative alkali or alkaline earth metal salts includesodium, lithium, potassium, calcium, magnesium, and the like. Furtherpharmaceutically acceptable salts include, when appropriate, nontoxicammonium, quaternary ammonium, and amine cations formed usingcounterions such as halide, hydroxide, carboxylate, sulfate, phosphate,nitrate, alkyl having from 1 to 6 carbon atoms, sulfonate and arylsulfonate.

As used herein, the term “pharmaceutically acceptable ester” refers toesters of the bifunctional compounds formed by the process of thepresent application which hydrolyze in vivo and include those that breakdown readily in the human body to leave the parent compound or a saltthereof. Suitable ester groups include, for example, those derived frompharmaceutically acceptable aliphatic carboxylic acids, particularlyalkanoic, alkenoic, cycloalkanoic and alkanedioic acids, in which eachalkyl or alkenyl moiety advantageously has not more than 6 carbon atoms.Examples of particular esters include, but are not limited to, formates,acetates, propionates, butyrates, acrylates and ethylsuccinates.

The term “pharmaceutically acceptable prodrugs” as used herein, refersto those prodrugs of the bifunctional compounds formed by the process ofthe present application which are, within the scope of sound medicaljudgment, suitable for use in contact with the tissues of humans andlower animals with undue toxicity, irritation, allergic response, andthe like, commensurate with a reasonable benefit/risk ratio, andeffective for their intended use, as well as the zwitterionic forms,where possible, of the compounds of the present application. “Prodrug”,as used herein, means a compound which is convertible in vivo bymetabolic means (e.g., by hydrolysis) to afford any compound delineatedby the formulae of the instant application. Various forms of prodrugsare known in the art, for example, as discussed in Bundgaard, (ed.),Design of Prodrugs, Elsevier (1985); Widder, et al. (ed.), Methods inEnzymology, vol. 4, Academic Press (1985); Krogsgaard-Larsen, et al.,(ed). “Design and Application of Prodrugs, Textbook of Drug Design andDevelopment, Chapter 5, 113-191 (1991); Bundgaard, et al., Journal ofDrug Deliver Reviews, 8:1-38(1992); Bundgaard, J. of PharmaceuticalSciences, 77:285 et seq. (1988); Higuchi and Stella (eds.) Prodrugs asNovel Drug Delivery Systems, American Chemical Society (1975); andBernard Testa & Joachim Mayer, “Hydrolysis In Drug And ProdrugMetabolism: Chemistry, Biochemistry And Enzymology,” John Wiley andSons, Ltd. (2002).

This application also encompasses pharmaceutical compositionscontaining, and methods of treating disorders through administering,pharmaceutically acceptable prodrugs of bifunctional compounds of theapplication. For example, compounds of the application having freeamino, amido, hydroxy or carboxylic groups can be converted intoprodrugs. Prodrugs include compounds wherein an amino acid residue, or apolypeptide chain of two or more (e.g., two, three or four) amino acidresidues is covalently joined through an amide or ester bond to a freeamino, hydroxy or carboxylic acid group of compounds of the application.The amino acid residues include but are not limited to the 20 naturallyoccurring amino acids commonly designated by three letter symbols andalso includes 4-hydroxyproline, hydroxylysine, demosine, isodemosine,3-methylhistidine, norvalin, beta-alanine, gamma-aminobutyric acid,citrulline, homocysteine, homoserine, ornithine and methionine sulfone.Additional types of prodrugs are also encompassed. For instance, freecarboxyl groups can be derivatized as amides or alkyl esters. Freehydroxy groups may be derivatized using groups including but not limitedto hemisuccinates, phosphate esters, dimethylaminoacetates, andphosphoryloxymethyloxy carbonyls, as outlined in Advanced Drug DeliveryReviews, 1996, 19, 1 15. Carbamate prodrugs of hydroxy and amino groupsare also included, as are carbonate prodrugs, sulfonate esters andsulfate esters of hydroxy groups. Derivatization of hydroxy groups as(acyloxy)methyl and (acyloxy)ethyl ethers wherein the acyl group may bean alkyl ester, optionally substituted with groups including but notlimited to ether, amine and carboxylic acid functionalities, or wherethe acyl group is an amino acid ester as described above, are alsoencompassed. Prodrugs of this type are described in J. Med. Chem. 1996,39, 10. Free amines can also be derivatized as amides, sulfonamides orphosphonamides. All of these prodrug moieties may incorporate groupsincluding but not limited to ether, amine and carboxylic acidfunctionalities.

The application also provides for a pharmaceutical compositioncomprising a therapeutically effective amount of a bifunctional compoundof the application, or an enantiomer, diastereomer, stereoisomer, orpharmaceutically acceptable salt thereof, and a pharmaceuticallyacceptable carrier.

In another aspect, the application provides a kit comprising abifunctional compound capable of inhibiting CDK8 activity selected fromone or more compounds disclosed herein, or a pharmaceutically acceptablesalt, hydrate, solvate, prodrug, stereoisomer, or tautomer thereof,optionally in combination with a second agent and instructions for usein treating cancer.

In another aspect, the application provides a method of synthesizing abifunctional compound disclosed herein.

The synthesis of the bifunctional compounds of the application can befound herein and in the Examples below.

Other embodiments are a method of making a bifunctional compound of anyof the formulae herein using any one, or combination of, reactionsdelineated herein. The method can include the use of one or moreintermediates or chemical reagents delineated herein.

Another aspect is an isotopically labeled bifunctional compound of anyof the formulae delineated herein. Such compounds have one or moreisotope atoms which may or may not be radioactive (e.g., ³H, ²H, ¹⁴C,¹³C, ¹⁸F, ³⁵S, ³²P, ¹²⁵I, and ¹³¹I) introduced into the bifunctionalcompound. Such compounds are useful for drug metabolism studies anddiagnostics, as well as therapeutic applications.

A bifunctional compound of the application can be prepared as apharmaceutically acceptable acid addition salt by reacting the free baseform of the compound with a pharmaceutically acceptable inorganic ororganic acid. Alternatively, a pharmaceutically acceptable base additionsalt of a bifunctional compound of the application can be prepared byreacting the free acid form of the bifunctional compound with apharmaceutically acceptable inorganic or organic base.

Alternatively, the salt forms of the bifunctional compounds of theapplication can be prepared using salts of the starting materials orintermediates.

The free acid or free base forms of the bifunctional compounds of theapplication can be prepared from the corresponding base addition salt oracid addition salt from, respectively. For example, a bifunctionalcompound of the application in an acid addition salt form can beconverted to the corresponding free base by treating with a suitablebase (e.g., ammonium hydroxide solution, sodium hydroxide, and thelike). A bifunctional compound of the application in a base additionsalt form can be converted to the corresponding free acid by treatingwith a suitable acid (e.g., hydrochloric acid, etc.).

Prodrugs of the bifunctional compounds of the application can beprepared by methods known to those of ordinary skill in the art (e.g.,for further details see Saulnier et al., (1994), Bioorganic andMedicinal Chemistry Letters, Vol. 4, p. 1985). For example, appropriateprodrugs can be prepared by reacting a non-derivatized bifunctionalcompound of the application with a suitable carbamylating agent (e.g.,1,1-acyloxyalkylcarbanochloridate, para-nitrophenyl carbonate, or thelike).

Protected derivatives of the bifunctional compounds of the applicationcan be made by means known to those of ordinary skill in the art. Adetailed description of techniques applicable to the creation ofprotecting groups and their removal can be found in T. W. Greene,“Protecting Groups in Organic Chemistry”, 3rd edition, John Wiley andSons, Inc., 1999.

Compounds of the present application can be conveniently prepared, orformed during the process of the application, as solvates (e.g.,hydrates). Hydrates of bifunctional compounds of the present applicationcan be conveniently prepared by recrystallization from anaqueous/organic solvent mixture, using organic solvents such as dioxin,tetrahydrofuran or methanol.

Acids and bases useful in the methods herein are known in the art. Acidcatalysts are any acidic chemical, which can be inorganic (e.g.,hydrochloric, sulfuric, nitric acids, aluminum trichloride) or organic(e.g., camphorsulfonic acid, p-toluenesulfonic acid, acetic acid,ytterbium triflate) in nature. Acids are useful in either catalytic orstoichiometric amounts to facilitate chemical reactions. Bases are anybasic chemical, which can be inorganic (e.g., sodium bicarbonate,potassium hydroxide) or organic (e.g., triethylamine, pyridine) innature. Bases are useful in either catalytic or stoichiometric amountsto facilitate chemical reactions.

Combinations of substituents and variables envisioned by thisapplication are only those that result in the formation of stablecompounds. The term “stable”, as used herein, refers to compounds whichpossess stability sufficient to allow manufacture and which maintainsthe integrity of the compound for a sufficient period of time to beuseful for the purposes detailed herein (e.g., therapeutic orprophylactic administration to a subject).

When any variable (e.g., R₁₄) occurs more than one time in anyconstituent or formula for a compound, its definition at each occurrenceis independent of its definition at every other occurrence. Thus, forexample, if a group is shown to be substituted with one or more R₁₄moieties, then R₁₄ at each occurrence is selected independently from thedefinition of R₁₄. Also, combinations of substituents and/or variablesare permissible, but only if such combinations result in stablecompounds within a designated atom's normal valency.

In addition, some of the compounds of this application have one or moredouble bonds, or one or more asymmetric centers. Such compounds canoccur as racemates, racemic mixtures, single enantiomers, individualdiastereomers, diastereomeric mixtures, and cis- or trans- or F- orZ-double isomeric forms, and other stereoisomeric forms that may bedefined, in terms of absolute stereochemistry, as (R)- or (S)-, or as(D)- or (L)- for amino acids. When the compounds described hereincontain olefinic double bonds or other centers of geometric asymmetry,and unless specified otherwise, it is intended that the compoundsinclude both E and Z geometric isomers. The configuration of anycarbon-carbon double bond appearing herein is selected for convenienceonly and is not intended to designate a particular configuration unlessthe text so states; thus a carbon-carbon double bond depictedarbitrarily herein as trans may be cis, trans, or a mixture of the twoin any proportion. All such isomeric forms of such compounds areexpressly included in the present application.

Optical isomers may be prepared from their respective optically activeprecursors by the procedures described herein, or by resolving theracemic mixtures. The resolution can be carried out in the presence of aresolving agent, by chromatography or by repeated crystallization or bysome combination of these techniques which are known to those skilled inthe art. Further details regarding resolutions can be found in Jacques,et al., Enantiomers, Racemates, and Resolutions (John Wiley & Sons,1981).

“Isomerism” means compounds that have identical molecular formulae butdiffer in the sequence of bonding of their atoms or in the arrangementof their atoms in space. Isomers that differ in the arrangement of theiratoms in space are termed “stereoisomers”. Stereoisomers that are notmirror images of one another are termed “diastereoisomers”, andstereoisomers that are non-superimposable mirror images of each otherare termed “enantiomers” or sometimes optical isomers. A mixturecontaining equal amounts of individual enantiomeric forms of oppositechirality is termed a “racemic mixture”.

A carbon atom bonded to four non-identical substituents is termed a“chiral center”.

“Chiral isomer” means a compound with at least one chiral center.Compounds with more than one chiral center may exist either as anindividual diastereomer or as a mixture of diastereomers, termed“diastereomeric mixture”. When one chiral center is present, astereoisomer may be characterized by the absolute configuration (R or S)of that chiral center. Absolute configuration refers to the arrangementin space of the substituents attached to the chiral center. Thesubstituents attached to the chiral center under consideration areranked in accordance with the Sequence Rule of Cahn, Ingold and Prelog.(Cahn et al., Angew. Chem. Inter. Edit. 1966, 5, 385; errata 511; Cahnet al., Angew. Chem. 1966, 78, 413; Cahn and Ingold, J. Chem. Soc. 1951(London), 612; Cahn et al., Experientia 1956, 12, 81; Cahn, J. Chem.Educ. 1964, 41, 116).

“Geometric isomer” means the diastereomers that owe their existence tohindered rotation about double bonds. These configurations aredifferentiated in their names by the prefixes cis and trans, or Z and E,which indicate that the groups are on the same or opposite side of thedouble bond in the molecule according to the Cahn-Ingold-Prelog rules.

Furthermore, the structures and other compounds discussed in thisapplication include all atropic isomers thereof. “Atropic isomers” are atype of stereoisomer in which the atoms of two isomers are arrangeddifferently in space. Atropic isomers owe their existence to arestricted rotation caused by hindrance of rotation of large groupsabout a central bond. Such atropic isomers typically exist as a mixture,however as a result of recent advances in chromatography techniques, ithas been possible to separate mixtures of two atropic isomers in selectcases.

“Tautomer” is one of two or more structural isomers that exist inequilibrium and is readily converted from one isomeric form to another.This conversion results in the formal migration of a hydrogen atomaccompanied by a switch of adjacent conjugated double bonds. Tautomersexist as a mixture of a tautomeric set in solution. In solid form,usually one tautomer predominates. In solutions where tautomerization ispossible, a chemical equilibrium of the tautomers will be reached. Theexact ratio of the tautomers depends on several factors, includingtemperature, solvent and pH. The concept of tautomers that areinterconvertable by tautomerizations is called tautomerism.

Of the various types of tautomerism that are possible, two are commonlyobserved. In keto-enol tautomerism a simultaneous shift of electrons anda hydrogen atom occurs. Ring-chain tautomerism arises as a result of thealdehyde group (—CHO) in a sugar chain molecule reacting with one of thehydroxy groups (—OH) in the same molecule to give it a cyclic(ring-shaped) form as exhibited by glucose. Common tautomeric pairs are:ketone-enol, amide-nitrile, lactam-lactim, amide-imidic acid tautomerismin heterocyclic rings (e.g., in nucleobases such as guanine, thymine andcytosine), amine-enamine and enamine-enamine. The compounds of thisapplication may also be represented in multiple tautomeric forms, insuch instances, the application expressly includes all tautomeric formsof the compounds described herein (e.g., alkylation of a ring system mayresult in alkylation at multiple sites, the application expresslyincludes all such reaction products).

In the present application, the structural formula of the bifunctionalcompound represents a certain isomer for convenience in some cases, butthe present application includes all isomers, such as geometricalisomers, optical isomers based on an asymmetrical carbon, stereoisomers,tautomers, and the like. In the present specification, the structuralformula of the compound represents a certain isomer for convenience insome cases, but the present application includes all isomers, such asgeometrical isomers, optical isomers based on an asymmetrical carbon,stereoisomers, tautomers, and the like.

Additionally, the compounds of the present application, for example, thesalts of the bifunctional compounds, can exist in either hydrated orunhydrated (the anhydrous) form or as solvates with other solventmolecules. Non-limiting examples of hydrates include monohydrates,dihydrates, etc. Non-limiting examples of solvates include ethanolsolvates, acetone solvates, etc.

“Solvate” means solvent addition forms that contain eitherstoichiometric or non stoichiometric amounts of solvent. Some compoundshave a tendency to trap a fixed molar ratio of solvent molecules in thecrystalline solid state, thus forming a solvate. If the solvent is waterthe solvate formed is a hydrate; and if the solvent is alcohol, thesolvate formed is an alcoholate. Hydrates are formed by the combinationof one or more molecules of water with one molecule of the substance inwhich the water retains its molecular state as H₂O.

The synthesized bifunctional compounds can be separated from a reactionmixture and further purified by a method such as column chromatography,high pressure liquid chromatography, or recrystallization. As can beappreciated by the skilled artisan, further methods of synthesizing thebifunctional compounds of the formulae herein will be evident to thoseof ordinary skill in the art. Additionally, the various synthetic stepsmay be performed in an alternate sequence or order to give the desiredcompounds. In addition, the solvents, temperatures, reaction durations,etc. delineated herein are for purposes of illustration only and one ofordinary skill in the art will recognize that variation of the reactionconditions can produce the desired bridged macrocyclic products of thepresent application. Synthetic chemistry transformations and protectinggroup methodologies (protection and deprotection) useful in synthesizingthe compounds described herein are known in the art and include, forexample, those such as described in R. Larock, Comprehensive OrganicTransformations, VCH Publishers (1989); T. W. Greene and P. G. M. Wuts,Protective Groups in Organic Synthesis, 2d. Ed., John Wiley and Sons(1991); L. Fieser and M. Fieser, Fieser and Fieser's Reagents forOrganic Synthesis, John Wiley and Sons (1994); and L. Paquette, ed.,Encyclopedia of Reagents for Organic Synthesis, John Wiley and Sons(1995), and subsequent editions thereof.

The compounds of this application may be modified by appending variousfunctionalities via any synthetic means delineated herein to enhanceselective biological properties. Such modifications are known in the artand include those which increase biological penetration into a givenbiological system (e.g., blood, lymphatic system, central nervoussystem), increase oral availability, increase solubility to allowadministration by injection, alter metabolism and alter rate ofexcretion.

The compounds of the application are defined herein by their chemicalstructures and/or chemical names. Where a compound is referred to byboth a chemical structure and a chemical name, and the chemicalstructure and chemical name conflict, the chemical structure isdeterminative of the compound's identity.

The recitation of a listing of chemical groups in any definition of avariable herein includes definitions of that variable as any singlegroup or combination of listed groups. The recitation of an embodimentfor a variable herein includes that embodiment as any single embodimentor in combination with any other embodiments or portions thereof.

Method of Synthesizing the Compounds

Compounds of the present application can be prepared in a variety ofways using commercially available starting materials, compounds known inthe literature, or from readily prepared intermediates, by employingstandard synthetic methods and procedures either known to those skilledin the art, or which will be apparent to the skilled artisan in light ofthe teachings herein. Standard synthetic methods and procedures for thepreparation of organic molecules and functional group transformationsand manipulations can be obtained from the relevant scientificliterature or from standard textbooks in the field. Although not limitedto any one or several sources, classic texts such as Smith, M. B.,March, J., March's Advanced Organic Chemistry: Reactions, Mechanisms,and Structure, 5^(th) edition, John Wiley & Sons: New York, 2001; andGreene, T. W., Wuts, P. G. M., Protective Groups in Organic Synthesis,3^(rd) edition, John Wiley & Sons: New York, 1999, incorporated byreference herein, are useful and recognized reference textbooks oforganic synthesis known to those in the art. The following descriptionsof synthetic methods are designed to illustrate, but not to limit,general procedures for the preparation of compounds of the presentapplication. The processes generally provide the desired final compoundat or near the end of the overall process, although it may be desirablein certain instances to further convert the compound to apharmaceutically acceptable salt, ester or prodrug thereof. Suitablesynthetic routes are depicted in the schemes below.

Those skilled in the art will recognize if a stereocenter exists in thecompounds disclosed herein. Accordingly, the present applicationincludes both possible stereoisomers (unless specified in the synthesis)and includes not only racemic compounds but the individual enantiomersand/or diastereomers as well. When a compound is desired as a singleenantiomer or diastereomer, it may be obtained by stereospecificsynthesis or by resolution of the final product or any convenientintermediate. Resolution of the final product, an intermediate, or astarting material may be affected by any suitable method known in theart. See, for example, “Stereochemistry of Organic Compounds” by E. L.Eliel, S. H. Wilen, and L. N. Mander (Wiley-Interscience, 1994).

The compounds of the present application can be prepared in a number ofways well known to those skilled in the art of organic synthesis. By wayof example, compounds of the present application can be synthesizedusing the methods described below, together with synthetic methods knownin the art of synthetic organic chemistry, or variations thereon asappreciated by those skilled in the art. Preferred methods include butare not limited to those methods described below.

Compounds of the present application can be synthesized by following thesteps outlined in General Scheme 1 and 2 which comprise differentsequences of assembling intermediates. Starting materials are eithercommercially available or made by known procedures in the reportedliterature or as illustrated.

wherein R₁₃, R₁₄, R₁₅, R₁₆, W, p1, q, and v are as defined herein above.

The general way of preparing representative compounds of the presentapplication (i.e., Compound of Formula (1) shown above) usingintermediates 1a, 1b, 1c, 1d, 1e, 1f, and 1g is outlined in GeneralScheme 1. Reaction of 1a with 1b in the presence of a base, i.e.,diisopropylethylamine (DIPEA), and in a solvent, i.e., dimethylformamide(DMF), provides intermediate 1c. Reaction of 1d with fluoride 1cprovides intermediate 1e. Deprotection of the 1e in the presence of TFAin a solvent, i.e., dichloromethane (DCM) or methanol (MeOH), provides1f. Coupling of 1f and Target Ligand 1g under standard couplingconditions using a coupling reagent, i.e.,1-ethyl-3-(3-dimethylaminopropyl) carbodiimide (EDC) andhydroxybenzotriazole, in a solvent, i.e., DCM or DMF, providesbifunctional compound of formula (I).

Biological AssaysCell Viability Assay

Wild-type or cereblon null cells are treated with various concentrationsof a bifunctional compound of the invention and allowed to grow. Cellsare then assayed to determine cell viability by measuring the amount ofATP present, which is an indicator of cell metabolic activity. Resultsare graphed as relative luminescent values.

Enzyme Degradation Assay

Cells are treated with a control or a bifunctional compound of theapplication alone or in combination with an agent that blocksproteasomal degradation at a single concentration or variousconcentrations. After treatment, cells are washed and harvested byresuspending in buffer and lysed on ice 30 minutes. Lysates are thencleared by centrifugation. Samples are boiled and equal amount ofprotein is loaded onto polyacrylamide gel. The gel is transferred tonitrocellulose and blotted for CDK8, mTOR, STAT1 serine 727phosphorylation or actin.

Western Blotting on CDK8

Cells are treated with a control or a bifunctional compound of theapplication at various concentrations for a desired period of time.Cells are then lysed in a suitable buffer. Protein concentration may bemeasured with any appropriate assay known in the art. Equivalent amountsof the samples are loaded on a polyacrylamide gel, transferred tonitrocellulose membranes, and immunoblotted with antibodies against CDK8and a loading control, such as actin. Labeled secondary antibodies areadded and washed. The signals from the label are detected.

Methods of the Application

In another aspect, the application provides a method of modulating akinase, comprising contacting the kinase with a bifunctional compounddisclosed herein, or a pharmaceutically acceptable salt, hydrate,solvate, prodrug, stereoisomer, or tautomer thereof, or with apharmaceutical composition disclosed herein. In some embodiments, thekinase is CDK8.

In another aspect, the application provides a method of inhibiting akinase, comprising contacting the kinase with a bifunctional compounddisclosed herein, or a pharmaceutically acceptable salt, hydrate,solvate, prodrug, stereoisomer, or tautomer thereof, or with apharmaceutical composition disclosed herein. In some embodiments, thekinase is CDK8.

In still another aspect, the application provides a method of inhibitingcyclin-dependent kinase (CDK8), the method comprising administering to asubject in need thereof an effective amount of a bifunctional compounddisclosed herein, or a pharmaceutically acceptable salt, hydrate,solvate, prodrug, stereoisomer, or tautomer thereof.

In still another aspect, the application provides a method of inhibitingcyclin-dependent kinase (CDK8), the method comprising administering to asubject in need thereof an effective amount of a pharmaceuticalcomposition comprising a bifunctional compound disclosed herein, or apharmaceutically acceptable salt, hydrate, solvate, prodrug,stereoisomer, or tautomer thereof and a pharmaceutically acceptablecarrier.

Another aspect of the application provides a method of treating orpreventing a disease, the method comprising administering to a subjectin need thereof an effective amount of a bifunctional compound disclosedherein, or a pharmaceutically acceptable salt, hydrate, solvate,prodrug, stereoisomer, or tautomer thereof. In some embodiments, thedisease is mediated by a kinase. In further embodiments, the kinase isCDK8.

Another aspect of the application provides a method of treating orpreventing a disease, the method comprising administering to a subjectin need thereof an effective amount of a pharmaceutical compositioncomprising a bifunctional compound disclosed herein, or apharmaceutically acceptable salt, hydrate, solvate, prodrug,stereoisomer, or tautomer thereof and a pharmaceutically acceptablecarrier. In some embodiments, the disease is mediated by a kinase. Infurther embodiments, the kinase is CDK8.

In some embodiments, the disease is mediated by CDK8 (e.g., CDK8 plays arole in the initiation or development of the disease).

In certain embodiments, the disease or disorder is cancer or aproliferation disease.

In further embodiments, the disease or disorder is lung cancer, coloncancer, breast cancer, prostate cancer, liver cancer, pancreas cancer,brain cancer, kidney cancer, ovarian cancer, stomach cancer, skincancer, bone cancer, gastric cancer, breast cancer, pancreatic cancer,glioma, glioblastoma, hepatocellular carcinoma, papillary renalcarcinoma, head and neck squamous cell carcinoma, leukemias, lymphomas,myelomas, or solid tumors.

In other embodiments, the disease or disorder is inflammation,arthritis, rheumatoid arthritis, spondyiarthropathies, gouty arthritis,osteoarthritis, juvenile arthritis, and other arthritic conditions,systemic lupus erythematosus (SLE), skin-related conditions, psoriasis,eczema, burns, dermatitis, neuroinflammation, allergy, pain, neuropathicpain, fever, pulmonary disorders, lung inflammation, adult respiratorydistress syndrome, pulmonary sarcoisosis, asthma, silicosis, chronicpulmonary inflammatory disease, and chronic obstructive pulmonarydisease (COPD), cardiovascular disease, arteriosclerosis, myocardialinfarction (including post-myocardial infarction indications),thrombosis, congestive heart failure, cardiac reperfusion injury, aswell as complications associated with hypertension and/or heart failuresuch as vascular organ damage, restenosis, cardiomyopathy, strokeincluding ischemic and hemorrhagic stroke, reperfusion injury, renalreperfusion injury, ischemia including stroke and brain ischemia, andischemia resulting from cardiac/coronary bypass, neurodegenerativedisorders, liver disease and nephritis, gastrointestinal conditions,inflammatory bowel disease, Crohn's disease, gastritis, irritable bowelsyndrome, ulcerative colitis, ulcerative diseases, gastric ulcers, viraland bacterial infections, sepsis, septic shock, gram negative sepsis,malaria, meningitis, HIV infection, opportunistic infections, cachexiasecondary to infection or malignancy, cachexia secondary to acquiredimmune deficiency syndrome (AIDS), AIDS, ARC (AIDS related complex),pneumonia, herpes virus, myalgias due to infection, influenza,autoimmune disease, graft vs. host reaction and allograft rejections,treatment of bone resorption diseases, osteoporosis, multiple sclerosis,cancer, leukemia, lymphoma, colorectal cancer, brain cancer, bonecancer, epithelial call-derived neoplasia (epithelial carcinoma), basalcell carcinoma, adenocarcinoma, gastrointestinal cancer, lip cancer,mouth cancer, esophageal cancer, small bowel cancer, stomach cancer,colon cancer, liver cancer, bladder cancer, pancreas cancer, ovariancancer, cervical cancer, lung cancer, breast cancer, skin cancer,squamous cell and/or basal cell cancers, prostate cancer, renal cellcarcinoma, and other known cancers that affect epithelial cellsthroughout the body, chronic myelogenous leukemia (CML), acute myeloidleukemia (AML) and acute promyelocytic leukemia (APL), angiogenesisincluding neoplasia, metastasis, central nervous system disorders,central nervous system disorders having an inflammatory or apoptoticcomponent, Alzheimer's disease, Parkinson's disease, Huntington'sdisease, amyotrophic lateral sclerosis, spinal cord injury, andperipheral neuropathy, or B-Cell Lymphoma.

In further embodiments, the disease or disorder is inflammation,arthritis, rheumatoid arthritis, spondyloarthropathies, gouty arthritis,osteoarthritis, juvenile arthritis, and other arthritic conditions,systemic lupus erythematosus (SLE), skin-related conditions, psoriasis,eczema, dermatitis, pain, pulmonary disorders, lung inflammation, adultrespiratory distress syndrome, pulmonary sarcoisosis, asthma, chronicpulmonary inflammatory disease, and chronic obstructive pulmonarydisease (COPD), cardiovascular disease, arteriosclerosis, myocardialinfarction (including post-myocardial infarction indications),congestive heart failure, cardiac reperfusion injury, inflammatory boweldisease, Crohn's disease, gastritis, irritable bowel syndrome, leukemiaor lymphoma.

Another aspect of the application provides a method of treating a kinasemediated disorder, the method comprising administering to a subject inneed thereof an effective amount of a bifunctional compound disclosedherein, or a pharmaceutically acceptable salt, hydrate, solvate,prodrug, stereoisomer, or tautomer thereof. In some embodiments, thebifunctional compound is an inhibitor of CDK8. In other embodiments, thesubject is administered an additional therapeutic agent. In otherembodiments, the bifunctional compound and the additional therapeuticagent are administered simultaneously or sequentially.

Another aspect of the application provides a method of treating a kinasemediated disorder, the method comprising administering to a subject inneed thereof an effective amount of a pharmaceutical compositioncomprising a bifunctional compound disclosed herein, or apharmaceutically acceptable salt, hydrate, solvate, prodrug,stereoisomer, or tautomer thereof and a pharmaceutically acceptablecarrier. In some embodiments, the bifunctional compound is an inhibitorof CDK8. In other embodiments, the subject is administered an additionaltherapeutic agent. In other embodiments, the pharmaceutical compositioncomprising a bifunctional compound and the additional therapeutic agentare administered simultaneously or sequentially.

In other embodiments, the disease or disorder is cancer. In furtherembodiments, the cancer is lung cancer, colon cancer, breast cancer,prostate cancer, liver cancer, pancreas cancer, brain cancer, kidneycancer, ovarian cancer, stomach cancer, skin cancer, bone cancer,gastric cancer, breast cancer, pancreatic cancer, glioma, glioblastoma,hepatocellular carcinoma, papillary renal carcinoma, head and necksquamous cell carcinoma, leukemias, lymphomas, myelomas, or solidtumors.

Another aspect of the present application relates to a method oftreating or preventing a proliferative disease. The method comprisesadministering to a subject in need thereof an effective amount of abifunctional compound of the application, or a pharmaceuticallyacceptable salt, hydrate, solvate, prodrug, stereoisomer, or tautomerthereof.

Another aspect of the present application relates to a method oftreating or preventing a proliferative disease. The method comprisesadministering to a subject in need thereof an effective amount of apharmaceutical composition comprising a bifunctional compound disclosedherein, or a pharmaceutically acceptable salt, hydrate, solvate,prodrug, stereoisomer, or tautomer thereof and a pharmaceuticallyacceptable carrier.

In another aspect, the application provides a method of treating orpreventing cancer, wherein the cancer cell comprises activated CDK8,comprising administering to a subject in need thereof an effectiveamount of a bifunctional compound disclosed herein, or apharmaceutically acceptable salt, hydrate, solvate, prodrug,stereoisomer, or tautomer thereof.

In another aspect, the application provides a method of treating orpreventing cancer, wherein the cancer cell comprises activated CDK8,comprising administering to a subject in need thereof an effectiveamount of a pharmaceutical composition comprising a bifunctionalcompound disclosed herein, or a pharmaceutically acceptable salt,hydrate, solvate, prodrug, stereoisomer, or tautomer thereof and apharmaceutically acceptable carrier.

In certain embodiments, the CDK8 activation is selected from mutation ofCDK8, amplification of CDK8, expression of CDK8, and ligand mediatedactivation of CDK8.

Another aspect of the application provides a method of treating orpreventing cancer in a subject, wherein the subject is identified asbeing in need of CDK8 inhibition for the treatment of cancer, comprisingadministering to the subject an effective amount of a bifunctionalcompound disclosed herein, or a pharmaceutically acceptable salt,hydrate, solvate, prodrug, stereoisomer, or tautomer thereof.

Another aspect of the application provides a method of treating orpreventing cancer in a subject, wherein the subject is identified asbeing in need of CDK8 inhibition for the treatment of cancer, comprisingadministering to the subject an effective amount of a pharmaceuticalcomposition comprising a bifunctional compound disclosed herein, or apharmaceutically acceptable salt, hydrate, solvate, prodrug,stereoisomer, or tautomer thereof and a pharmaceutically acceptablecarrier.

In certain embodiments, the application provides a method of treatingany of the disorders described herein, wherein the subject is a human.In certain embodiments, the application provides a method of preventingany of the disorders described herein, wherein the subject is a human.

In another aspect, the application provides a bifunctional compounddisclosed herein, or a pharmaceutically acceptable salt, hydrate,solvate, prodrug, stereoisomer, or tautomer thereof, for use in themanufacture of a medicament for treating or preventing a disease inwhich CDK8 plays a role.

In still another aspect, the application provides a bifunctionalcompound of the application, or a pharmaceutically acceptable salt,hydrate, solvate, prodrug, stereoisomer, or tautomer thereof for use intreating or preventing a disease in which CDK8 plays a role.

In another aspect, the application provides a pharmaceutical compositioncomprising a bifunctional compound disclosed herein, or apharmaceutically acceptable salt, hydrate, solvate, prodrug,stereoisomer, or tautomer thereof and a pharmaceutically acceptablecarrier, for use in the manufacture of a medicament for treating orpreventing a disease in which CDK8 plays a role.

In still another aspect, the application provides a pharmaceuticalcomposition comprising a bifunctional compound disclosed herein, or apharmaceutically acceptable salt, hydrate, solvate, prodrug,stereoisomer, or tautomer thereof and a pharmaceutically acceptablecarrier, for use in treating or preventing a disease in which CDK8 playsa role.

As inhibitors of CDK8 kinase, the bifunctional compounds andcompositions of this application are particularly useful for treating orlessening the severity of a disease, condition, or disorder where aprotein kinase is implicated in the disease, condition, or disorder. Inone aspect, the present application provides a method for treating orlessening the severity of a disease, condition, or disorder where aprotein kinase is implicated in the disease state. In another aspect,the present application provides a method for treating or lessening theseverity of a kinase disease, condition, or disorder where inhibition ofenzymatic activity is implicated in the treatment of the disease. Inanother aspect, this application provides a method for treating orlessening the severity of a disease, condition, or disorder withbifunctional compounds that inhibit enzymatic activity by binding to theprotein kinase. Another aspect provides a method for treating orlessening the severity of a kinase disease, condition, or disorder byinhibiting enzymatic activity of the kinase with a protein kinaseinhibitor.

In some embodiments, said method is used to treat or prevent a conditionselected from autoimmune diseases, inflammatory diseases, proliferativeand hyperproliferative diseases, immunologically-mediated diseases, bonediseases, metabolic diseases, neurological and neurodegenerativediseases, cardiovascular diseases, hormone related diseases, allergies,asthma, and Alzheimer's disease. In other embodiments, said condition isselected from a proliferative disorder and a neurodegenerative disorder.

One aspect of this application provides bifunctional compounds that areuseful for the treatment of diseases, disorders, and conditionscharacterized by excessive or abnormal cell proliferation. Such diseasesinclude, but are not limited to, a proliferative or hyperproliferativedisease, and a neurodegenerative disease. Examples of proliferative andhyperproliferative diseases include, without limitation, cancer. Theterm “cancer” includes, but is not limited to, the following cancers:breast; ovary; cervix; prostate; testis, genitourinary tract; esophagus;larynx, glioblastoma; neuroblastoma; stomach; skin, keratoacanthoma;lung, epidermoid carcinoma, large cell carcinoma, small cell carcinoma,lung adenocarcinoma; bone; colon; colorectal; adenoma; pancreas,adenocarcinoma; thyroid, follicular carcinoma, undifferentiatedcarcinoma, papillary carcinoma; seminoma; melanoma; sarcoma; bladdercarcinoma; liver carcinoma and biliary passages; kidney carcinoma;myeloid disorders; lymphoid disorders, Hodgkin's, hairy cells; buccalcavity and pharynx (oral), lip, tongue, mouth, pharynx; small intestine;colonrectum, large intestine, rectum, brain and central nervous system;chronic myeloid leukemia (CML), and leukemia. The term “cancer”includes, but is not limited to, the following cancers: myeloma,lymphoma, or a cancer selected from gastric, renal, or and the followingcancers: head and neck, oropharangeal, non-small cell lung cancer(NSCLC), endometrial, hepatocarcinoma, Non-Hodgkins lymphoma, andpulmonary.

The term “cancer” refers to any cancer caused by the proliferation ofmalignant neoplastic cells, such as tumors, neoplasms, carcinomas,sarcomas, leukemias, lymphomas and the like. For example, cancersinclude, but are not limited to, mesothelioma, leukemias and lymphomassuch as cutaneous T-cell lymphomas (CTCL), noncutaneous peripheralT-cell lymphomas, lymphomas associated with human T-cell lymphotrophicvirus (HTLV) such as adult T-cell leukemia/lymphoma (ATLL), B-celllymphoma, acute nonlymphocytic leukemias, chronic lymphocytic leukemia,chronic myelogenous leukemia, acute myelogenous leukemia, lymphomas, andmultiple myeloma, non-Hodgkin lymphoma, acute lymphatic leukemia (ALL),chronic lymphatic leukemia (CLL), Hodgkin's lymphoma, Burkitt lymphoma,adult T-cell leukemia lymphoma, acute-myeloid leukemia (AML), chronicmyeloid leukemia (CML), or hepatocellular carcinoma. Further examplesinclude myelodisplastic syndrome, childhood solid tumors such as braintumors, neuroblastoma, retinoblastoma, Wilms' tumor, bone tumors, andsoft-tissue sarcomas, common solid tumors of adults such as head andneck cancers (e.g., oral, laryngeal, nasopharyngeal and esophageal),genitourinary cancers (e.g., prostate, bladder, renal, uterine, ovarian,testicular), lung cancer (e.g., small-cell and non-small cell), breastcancer, pancreatic cancer, melanoma and other skin cancers, stomachcancer, brain tumors, tumors related to Gorlin's syndrome (e.g.,medulloblastoma, meningioma, etc.), and liver cancer. Additionalexemplary forms of cancer which may be treated by the subjectbifunctional compounds include, but are not limited to, cancer ofskeletal or smooth muscle, stomach cancer, cancer of the smallintestine, rectum carcinoma, cancer of the salivary gland, endometrialcancer, adrenal cancer, anal cancer, rectal cancer, parathyroid cancer,and pituitary cancer.

Additional cancers that the bifunctional compounds described herein maybe useful in preventing, treating and studying are, for example, coloncarcinoma, familiary adenomatous polyposis carcinoma and hereditarynon-polyposis colorectal cancer, or melanoma. Further, cancers include,but are not limited to, labial carcinoma, larynx carcinoma, hypopharynxcarcinoma, tongue carcinoma, salivary gland carcinoma, gastriccarcinoma, adenocarcinoma, thyroid cancer (medullary and papillarythyroid carcinoma), renal carcinoma, kidney parenchyma carcinoma, cervixcarcinoma, uterine corpus carcinoma, endometrium carcinoma, chorioncarcinoma, testis carcinoma, urinary carcinoma, melanoma, brain tumorssuch as glioblastoma, astrocytoma, meningioma, medulloblastoma andperipheral neuroectodermal tumors, gall bladder carcinoma, bronchialcarcinoma, multiple myeloma, basalioma, teratoma, retinoblastoma,choroidea melanoma, seminoma, rhabdomyosarcoma, craniopharyngeoma,osteosarcoma, chondrosarcoma, myosarcoma, liposarcoma, fibrosarcoma,Ewing sarcoma, and plasmocytoma. In one aspect of the application, thepresent application provides for the use of one or more bifunctionalcompounds of the application in the manufacture of a medicament for thetreatment of cancer, including without limitation the various types ofcancer disclosed herein.

In some embodiments, the bifunctional compounds of this application areuseful for treating cancer, such as colorectal, thyroid, breast, andlung cancer; and myeloproliferative disorders, such as polycythemiavera, thrombocythemia, myeloid metaplasia with myelofibrosis, chronicmyelogenous leukemia, chronic myelomonocytic leukemia, hypereosinophilicsyndrome, juvenile myelomonocytic leukemia, and systemic mast celldisease. In some embodiments, the bifunctional compounds of thisapplication are useful for treating hematopoietic disorders, inparticular, acute-myelogenous leukemia (AML), chronic-myelogenousleukemia (CML), acute-promyelocytic leukemia, and acute lymphocyticleukemia (ALL).

This application further embraces the treatment or prevention of cellproliferative disorders such as hyperplasias, dysplasias andpre-cancerous lesions. Dysplasia is the earliest form of pre-cancerouslesion recognizable in a biopsy by a pathologist. The subjectbifunctional compounds may be administered for the purpose of preventingsaid hyperplasias, dysplasias or pre-cancerous lesions from continuingto expand or from becoming cancerous. Examples of pre-cancerous lesionsmay occur in skin, esophageal tissue, breast and cervicalintra-epithelial tissue.

Examples of neurodegenerative diseases include, without limitation,Adrenoleukodystrophy (ALD), Alexander's disease, Alper's disease,Alzheimer's disease, Amyotrophic lateral sclerosis (Lou Gehrig'sDisease), Ataxia telangiectasia, Batten disease (also known asSpielmeyer-Vogt-Sjogren-Batten disease), Bovine spongiformencephalopathy (BSE), Canavan disease, Cockayne syndrome, Corticobasaldegeneration, Creutzfeldt-Jakob disease, Familial fatal insomnia,Frontotemporal lobar degeneration, Huntington's disease, HIV-associateddementia, Kennedy's disease, Krabbe's disease, Lewy body dementia,Neuroborreliosis, Machado-Joseph disease (Spinocerebellar ataxia type3), Multiple System Atrophy, Multiple sclerosis, Narcolepsy, NiemannPick disease, Parkinson's disease, Pelizaeus-Merzbacher Disease, Pick'sdisease, Primary lateral sclerosis, Prion diseases, ProgressiveSupranuclear Palsy, Refsum's disease, Sandhoff disease, Schilder'sdisease, Subacute combined degeneration of spinal cord secondary toPernicious Anaemia, Spielmeyer-Vogt-Sjogren-Batten disease (also knownas Batten disease), Spinocerebellar ataxia (multiple types with varyingcharacteristics), Spinal muscular atrophy, Steele-Richardson-Olszewskidisease, Tabes dorsalis, and Toxic encephalopathy.

Another aspect of this application provides a method for the treatmentor lessening the severity of a disease selected from a proliferative orhyperproliterative disease, or a neurodegenerative disease, comprisingadministering an effective amount of a bifunctional compound, or apharmaceutically acceptable composition comprising a bifunctionalcompound, to a subject in need thereof.

As inhibitors of CDK8 kinase, the compounds and compositions of thisapplication are also useful in biological samples. One aspect of theapplication relates to inhibiting protein kinase activity in abiological sample, which method comprises contacting said biologicalsample with a bifunctional compound of the application or a compositioncomprising said bifunctional compound. The term “biological sample”, asused herein, means an in vitro or an ex vivo sample, including, withoutlimitation, cell cultures or extracts thereof; biopsied materialobtained from a mammal or extracts thereof; and blood, saliva, urine,feces, semen, tears, or other body fluids or extracts thereof.Inhibition of protein kinase activity in a biological sample is usefulfor a variety of purposes that are known to one of skill in the art.Examples of such purposes include, but are not limited to, bloodtransfusion, organ-transplantation, and biological specimen storage.

Another aspect of this application relates to the study of CDK8 kinasein biological and pathological phenomena; the study of intracellularsignal transduction pathways mediated by such protein kinases; and thecomparative evaluation of new protein kinase inhibitors. Examples ofsuch uses include, but are not limited to, biological assays such asenzyme assays and cell-based assays.

The activity of the compounds and compositions of the presentapplication as CDK8 inhibitors may be assayed in vitro, in vivo, or in acell line. In vitro assays include assays that determine inhibition ofeither the kinase activity or ATPase activity of the activated kinase.Alternate in vitro assays quantitate the ability of the inhibitor tobind to the protein kinase and may be measured either by radio labellingthe inhibitor prior to binding, isolating the inhibitor/kinase complexand determining the amount of radio label bound, or by running acompetition experiment where new inhibitors are incubated with thekinase bound to known radioligands. Detailed conditions for assaying acompound utilized in this application as an inhibitor of various kinasesare set forth in the Examples below.

In accordance with the foregoing, the present application furtherprovides a method for preventing or treating any of the diseases ordisorders described above in a subject in need of such treatment, whichmethod comprises administering to said subject a therapeuticallyeffective amount of a bifunctional compound of the application, or apharmaceutically acceptable salt, hydrate, solvate, prodrug,stereoisomer, or tautomer thereof. For any of the above uses, therequired dosage will vary depending on the mode of administration, theparticular condition to be treated and the effect desired.

Pharmaceutical Compositions

In another aspect, the application provides a pharmaceutical compositioncomprising a therapeutically effective amount of a bifunctional compoundof the present application or an enantiomer, diastereomer, stereoisomer,or pharmaceutically acceptable salt thereof, and a pharmaceuticallyacceptable carrier.

Bifunctional compounds of the application can be administered aspharmaceutical compositions by any conventional route, in particularenterally, e.g., orally, e.g., in the form of tablets or capsules, orparenterally, e.g., in the form of injectable solutions or suspensions,or topically, e.g., in the form of lotions, gels, ointments or creams,or in a nasal or suppository form. Pharmaceutical compositionscomprising a compound of the present application in free form or in apharmaceutically acceptable salt form in association with at least onepharmaceutically acceptable carrier or diluent can be manufactured in aconventional manner by mixing, granulating or coating methods. Forexample, oral compositions can be tablets or gelatin capsules comprisingthe active ingredient together with a) diluents, e.g., lactose,dextrose, sucrose, mannitol, sorbitol, cellulose and/or glycine; b)lubricants, e.g., silica, talcum, stearic acid, its magnesium or calciumsalt and/or polyethyleneglycol; for tablets also c) binders, e.g.,magnesium aluminum silicate, starch paste, gelatin, tragacanth,methylcellulose, sodium carboxymethylcellulose and orpolyvinylpyrrolidone; if desired d) disintegrants, e.g., starches, agar,alginic acid or its sodium salt, or effervescent mixtures; and/or e)absorbents, colorants, flavors and sweeteners. Injectable compositionscan be aqueous isotonic solutions or suspensions, and suppositories canbe prepared from fatty emulsions or suspensions. The compositions may besterilized and/or contain adjuvants, such as preserving, stabilizing,wetting or emulsifying agents, solution promoters, salts for regulatingthe osmotic pressure and/or buffers. In addition, they may also containother therapeutically valuable substances. Suitable formulations fortransdermal applications include an effective amount of a compound ofthe present application with a carrier. A carrier can include absorbablepharmacologically acceptable solvents to assist passage through the skinof the host. For example, transdermal devices are in the form of abandage comprising a backing member, a reservoir containing the compoundoptionally with carriers, optionally a rate controlling barrier todeliver the compound to the skin of the host at a controlled andpredetermined rate over a prolonged period of time, and means to securethe device to the skin. Matrix transdermal formulations may also beused. Suitable formulations for topical application, e.g., to the skinand eyes, are preferably aqueous solutions, ointments, creams or gelswell-known in the art. Such may contain solubilizers, stabilizers,tonicity enhancing agents, buffers and preservatives.

The pharmaceutical compositions of the present application comprise atherapeutically effective amount of a compound of the presentapplication formulated together with one or more pharmaceuticallyacceptable carriers. As used herein, the term “pharmaceuticallyacceptable carrier” means a non-toxic, inert solid, semi-solid or liquidfiller, diluent, encapsulating material or formulation auxiliary of anytype. Some examples of materials which can serve as pharmaceuticallyacceptable carriers include, but are not limited to, ion exchangers,alumina, aluminum stearate, lecithin, serum proteins, such as humanserum albumin, buffer substances such as phosphates, glycine, sorbicacid, or potassium sorbate, partial glyceride mixtures of saturatedvegetable fatty acids, water, salts or electrolytes, such as protaminesulfate, disodium hydrogen phosphate, potassium hydrogen phosphate,sodium chloride, zinc salts, colloidal silica, magnesium trisilicate,polyvinyl pyrrolidone, polyacrylates, waxes, polyethylenepolyoxypropylene-block polymers, wool fat, sugars such as lactose, glucose andsucrose; starches such as corn starch and potato starch; cellulose andits derivatives such as sodium carboxymethyl cellulose, ethyl celluloseand cellulose acetate; powdered tragacanth; malt; gelatin; talc;excipients such as cocoa butter and suppository waxes, oils such aspeanut oil, cottonseed oil; safflower oil; sesame oil; olive oil; cornoil and soybean oil; glycols such a propylene glycol or polyethyleneglycol; esters such as ethyl oleate and ethyl laurate, agar; bufferingagents such as magnesium hydroxide and aluminum hydroxide; alginic acid;pyrogen-free water, isotonic saline; Ringer's solution; ethyl alcohol,and phosphate buffer solutions, as well as other non-toxic compatiblelubricants such as sodium lauryl sulfate and magnesium stearate, as wellas coloring agents, releasing agents, coating agents, sweetening,flavoring and perfuming agents, preservatives and antioxidants can alsobe present in the composition, according to the judgment of theformulator.

The pharmaceutical compositions of this application can be administeredto humans and other animals orally, rectally, parenterally,intracisternally, intravaginally, intraperitoneally, topically (as bypowders, ointments, or drops), buccally, or as an oral or nasal spray.

Liquid dosage forms for oral administration include pharmaceuticallyacceptable emulsions, microemulsions, solutions, suspensions, syrups andelixirs. In addition to the active compounds, the liquid dosage formsmay contain inert diluents commonly used in the art such as, forexample, water or other solvents, solubilizing agents and emulsifierssuch as ethyl alcohol, isopropyl alcohol, ethyl carbonate, ethylacetate, benzyl alcohol, benzyl benzoate, propylene glycol, 1,3-butyleneglycol, dimethylformamide, oils (in particular, cottonseed, groundnut,corn, germ, olive, castor, and sesame oils), glycerol,tetrahydrofurfuryl alcohol, polyethylene glycols and fatty acid estersof sorbitan, and mixtures thereof. Besides inert diluents, the oralcompositions can also include adjuvants such as wetting agents,emulsifying and suspending agents, sweetening, flavoring, and perfumingagents.

Injectable preparations, for example, sterile injectable aqueous, oroleaginous suspensions may be formulated according to the known artusing suitable dispersing or wetting agents and suspending agents. Thesterile injectable preparation may also be a sterile injectablesolution, suspension or emulsion in a nontoxic parenterally acceptablediluent or solvent, for example, as a solution in 1,3-butanediol. Amongthe acceptable vehicles and solvents that may be employed are water,Ringer's solution, U.S.P. and isotonic sodium chloride solution. Inaddition, sterile, fixed oils are conventionally employed as a solventor suspending medium. For this purpose any bland fixed oil can beemployed including synthetic mono- or diglycerides. In addition, fattyacids such as oleic acid are used in the preparation of injectables.

In order to prolong the effect of a drug, it is often desirable to slowthe absorption of the drug from subcutaneous or intramuscular injection.This may be accomplished by the use of a liquid suspension ofcrystalline or amorphous material with poor water solubility. The rateof absorption of the drug then depends upon its rate of dissolutionwhich, in turn, may depend upon crystal size and crystalline form.Alternatively, delayed absorption of a parenterally administered drugform is accomplished by dissolving or suspending the drug in an oilvehicle.

Compositions for rectal or vaginal administration are preferablysuppositories which can be prepared by mixing the compounds of thisapplication with suitable non-irritating excipients or carriers such ascocoa butter, polyethylene glycol or a suppository wax which are solidat ambient temperature but liquid at body temperature and therefore meltin the rectum or vaginal cavity and release the active compound.

Solid compositions of a similar type may also be employed as fillers insoft and hard filled gelatin capsules using such excipients as lactoseor milk sugar as well as high molecular weight polyethylene glycols andthe like.

The active compounds can also be in micro-encapsulated form with one ormore excipients as noted above. The solid dosage forms of tablets,dragees, capsules, pills, and granules can be prepared with coatings andshells such as enteric coatings, release controlling coatings and othercoatings well known in the pharmaceutical formulating art. In such soliddosage forms the active compound may be admixed with at least one inertdiluent such as sucrose, lactose or starch. Such dosage forms may alsocomprise, as is normal practice, additional substances other than inertdiluents, e.g., tableting lubricants and other tableting aids such amagnesium stearate and microcrystalline cellulose. In the case ofcapsules, tablets and pills, the dosage forms may also comprisebuffering agents.

Dosage forms for topical or transdermal administration of a compound ofthis application include ointments, pastes, creams, lotions, gels,powders, solutions, sprays, inhalants or patches. The active componentis admixed under sterile conditions with a pharmaceutically acceptablecarrier and any needed preservatives or buffers as may be required.Ophthalmic formulation, ear drops, eye ointments, powders and solutionsare also contemplated as being within the scope of this application.

The ointments, pastes, creams and gels may contain, in addition to anactive compound of this application, excipients such as animal andvegetable fats, oils, waxes, paraffins, starch, tragacanth, cellulosederivatives, polyethylene glycols, silicones, bentonites, silicic acid,talc and zinc oxide, or mixtures thereof.

Powders and sprays can contain, in addition to the compounds of thisapplication, excipients such as lactose, talc, silicic acid, aluminumhydroxide, calcium silicates and polyamide powder, or mixtures of thesesubstances. Sprays can additionally contain customary propellants suchas chlorofluorohydrocarbons.

Transdermal patches have the added advantage of providing controlleddelivery of a compound to the body. Such dosage forms can be made bydissolving or dispensing the compound in the proper medium. Absorptionenhancers can also be used to increase the flux of the compound acrossthe skin. The rate can be controlled by either providing a ratecontrolling membrane or by dispersing the compound in a polymer matrixor gel.

Compounds and compositions of the application can be administered intherapeutically effective amounts in a combinational therapy with one ormore therapeutic agents (pharmaceutical combinations) or modalities,e.g., an anti-proliferative, anti-cancer, immunomodulatory oranti-inflammatory agent. Where the compounds of the application areadministered in conjunction with other therapies, dosages of theco-administered compounds will of course vary depending on the type ofco-drug employed, on the specific drug employed, on the condition beingtreated and so forth. Compounds and compositions of the application canbe administered in therapeutically effective amounts in a combinationaltherapy with one or more therapeutic agents (pharmaceuticalcombinations) or modalities, e.g., anti-proliferative, anti-cancer,immunomodulatory or anti-inflammatory agent, and/or non-drug therapies,etc. For example, synergistic effects can occur with anti-proliferative,anti-cancer, immunomodulatory or anti-inflammatory substances. Where thecompounds of the application are administered in conjunction with othertherapies, dosages of the co-administered compounds will of course varydepending on the type of co-drug employed, on the specific drugemployed, on the condition being treated and so forth.

Combination therapy includes the administration of the subject compoundsin further combination with one or more other biologically activeingredients (such as, but not limited to, a second CDK8 inhibitor, asecond and different antineoplastic agent, a second cyclin-dependentkinase inhibitor (i.e., CDK1, CDK2, CDK4, CDK6, CDK7, CDK9, CDK11,CDK12, CDK13, CDK14, etc.) and non-drug therapies (such as, but notlimited to, surgery or radiation treatment). For instance, the compoundsof the application can be used in combination with otherpharmaceutically active compounds, preferably compounds that are able toenhance the effect of the compounds of the application. The compounds ofthe application can be administered simultaneously (as a singlepreparation or separate preparation) or sequentially to the other drugtherapy or treatment modality. In general, a combination therapyenvisions administration of two or more drugs during a single cycle orcourse of therapy.

In another aspect of the application, the compounds may be administeredin combination with one or more separate pharmaceutical agents, e.g., achemotherapeutic agent, an immunotherapeutic agent, or an adjunctivetherapeutic agent.

EXAMPLES

Analytical Methods, Materials, and Instrumentation

All reactions were monitored Waters Acquity UPLC/MS system (Waters PDAeλ Detector, QDa Detector, Sample manager—FL, Binary Solvent Manager)using Acquity UPLC® BEH C18 column (2.1×50 mm, 1.7 μm particle size):solvent gradient=90% A at 0 min, 1% A at 1.8 min; solvent A=0.1% formicacid in Water; solvent B=0.1% formic acid in Acetonitrile; flow rate:0.6 mL/min. Reaction products were purified by flash columnchromatography using CombiFlash® Rf with Teledyne Isco RediSep® Rf HighPerformance Gold or Silicycle SiliaSep™ High Performance columns (4 g,12 g, 24 g, 40 g, or 80 g), Waters HPLC system using SunFire™ Prep C18column (19×100 mm, 5 μm particle size): solvent gradient=80% A at 0 min,5% A at 25 min; solvent A=0.035% TFA in Water; solvent B=0.035% TFA inMeOH; flow rate: 25 mL/min (Method A), and Waters Acquity UPLC/MS system(Waters PDA eλ Detector, QDa Detector, Sample manager—FL, Binary SolventManager) using Acquity UPLC® BEH C18 column (2.1×50 mm, 1.7 m particlesize): solvent gradient=80% A at 0 min, 5% A at 2 min; solvent A=0.1%formic acid in Water; solvent B=0.1% formic acid in Acetonitrile; flowrate: 0.6 mL/min (method B). The purity of all compounds was over 95%and was analyzed with Waters LC/MS system. ¹H NMR was obtained using a500 MHz Bruker Avance III. Chemical shifts are reported relative todimethyl sulfoxide (δ=2.50) for ¹H NMR. Data are reported as (br=broad,s=singlet, d=doublet, t=triplet, q=quartet, m=multiplet).

Abbreviations used in the following examples and elsewhere herein are:

-   -   atm atmosphere    -   br broad    -   DCM dichloromethane    -   DIEA N,N-diisopropylethylamine    -   DMA N,N-dimethylacetamide    -   DMF N,N-dimethylformamide    -   DMSO dimethyl sulfoxide    -   EDCI 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide    -   ESI electrospray ionization    -   EtOAc ethyl acetate    -   HCl hydrochloric acid    -   h hour(s)    -   HATU        bis(dimethylamino)methylene]-1H-1,2,3-triazolo[4,5-b]pyridinium        3-oxide hexafluoro-phosphate    -   HPLC high-performance liquid chromatography    -   LCMS liquid chromatography-mass spectrometry    -   m multiplet    -   MeOH methanol    -   MHz megahertz    -   min minutes    -   MS mass spectrometry    -   NMR nuclear magnetic resonance    -   Pd₂(dba)₃ tris(dibenzylideneacetone)dipalladium(0)    -   ppm parts per million    -   TBAF tetra-n-butylammonium fluoride    -   THF tetrahydrofuran    -   TLC thin layer chromatography    -   Xphos 2-dicyclohexylphosphino-2′,4′,6′-triisopropylbiphenyl

Example 1: Synthesis of2-(2,6-dioxopiperidin-3-yl)-4-((2-(3-(4-(4-(9-(isoquinolin-7-yl)-2-oxobenzo[h][1,6]naphthyridin-1(2H)-yl)-2-(trifluoromethyl)phenyl)piperazin-1-yl)-3-oxopropoxy)ethyl)amino)isoindoline-1,3-dione(I-1)

Step 1: Synthesis of Ethyl4-((4-(4-(tert-butoxycarbonyl)piperazin-1-yl)-3-(trifluoromethyl)phenyl)amino)-6-chloroquinoline-3-carboxylate

Ethyl 4,6-dichloroquinoline-3-carboxylate (569 mg, 2.1 mmol), tert-butyl4-(4-amino-2-(trifluoromethyl)phenyl)piperazine-1-carboxylate (727 mg,2.1 mmol), and DIEA (258 mg, 0.35 mL, 2 mmol) were mixed in dioxane (10mL). The mixture was stirred at 100° C. overnight. The reaction wasconcentrated and purified by flash column chromatography on silica gel(0-70% EtOAc in hexane) to give the title compound as a yellow solid(465 mg, 38%). LCMS: m/z 579.2 [M+1].

Step 2: Synthesis of tert-Butyl4-(4-(9-chloro-2-oxobenzo[h][1,6]naphthyridin-1(2H)-yl)-2-(trifluoromethyl)phenyl)piperazine-1-carboxylate

To a solution of ethyl4-((4-(4-(tert-butoxycarbonyl)piperazin-1-yl)-3-(trifluoromethyl)phenyl)amino)-6-chloroquinoline-3-carboxylate(465 mg, 0.80 mmol) in EtOH (10 mL) was added NaBH₄ (304 mg, 8.0 mmol)slowly. The mixture was stirred at rt overnight. The reaction wasconcentrated and extracted with EtOAc from H₂O, washed with brine, dried(Na₂SO₄), and concentrated. The residue was mixed in DCM (10 mL) andMnO₂ (2 g) was added. The mixture was stirred at rt overnight. Themixture was filtered through Celite and concentrated. The residue wasdissolved in EtOH (10 mL) and triethyl phosphonoacetate (362 mg, 1.6mmol) was added, followed by K₂CO₃ (331 mg, 2.4 mmol). The mixture wasstirred at 100° C. overnight. The mixture was concentrated and purifiedby flash column chromatography on silica gel (100% EtOAc) to give thetitle compound as a yellow solid (138 mg, 31% over three steps). LCMS:m/z 559.2 [M+1].

Step 3: Synthesis of9-(Isoquinolin-7-yl)-1-(4-(piperazin-1-yl)-3-(trifluoromethyl)phenyl)benzo[h][1,6]naphthyridin-2(1H)-one

To a sealed tube was added tert-Butyl4-(4-(9-chloro-2-oxobenzo[h][1,6]naphthyridin-1(2H)-yl)-2-(trifluoromethyl)phenyl)piperazine-1-carboxylate(138 mg, 0.25 mmol), isoquinolin-7-ylboronic acid (86.5 mg, 0.5 mmol),tBuXPhos (10.6 mg, 0.025 mmol), dioxane (6 mL), and Na₂CO₃ (1.5 mL). Themixture was degassed and Pd(PPh₃)₂CH₂ (17.5 mg, 0.025 mmol) was added.The mixture was stirred at 80° C. overnight. The mixture was filteredand concentrated. The residue was dissolved in DCM (1 mL) and TFA (1 mL)and stirred at rt for 1 h. The reaction was concentrated and purified byflash column chromatography on silica gel (0-10% MeOH in DCM) to givethe title compound as a yellow solid (131 mg, 95% over two steps). LCMS:m/z 552.2 [M+1].

Step 4: Synthesis of2-(2,6-Dioxopiperidin-3-yl)-4-((2-(3-(4-(4-(9-(isoquinolin-7-yl)-2-oxobenzo[h][1,6]naphthyridin-1(2H)-yl)-2-(trifluoromethyl)phenyl)piperazin-1-yl)-3-oxopropoxy)ethyl)amino)isoindoline-1,3-dione(I-1)

To a solution of9-(isoquinolin-7-yl)-1-(4-(piperazin-1-yl)-3-(trifluoromethyl)phenyl)benzo[h][1,6]naphthyridin-2(1H)-one(13 mg, 0.0232 mmol) and3-(2-((2-(2,6-dioxopiperidin-3-yl)-1,3-dioxoisoindolin-4-yl)amino)ethoxy)propanoicacid (13 mg, 0.0232 mmol) in DMF (0.5 mL) were added EDC (5.3 mg, 0.0278mmol), HOBT (4.1 mg, 0.03 mmol), and TEA (12 mg, 16 μL, 0.116 mmol). Themixture was stirred at rt overnight. The mixture was purified by reversephase HPLC (0-100% MeOH in H₂O) to give compound I-1 as a yellow solid(11 mg, 51%). ¹H NMR (500 MHz, DMSO-d₆) δ 11.01 (s, 1H), 9.43 (s, 1H),9.16 (s, 1H), 8.54 (d, J=6.0 Hz, 1H), 8.30 (d, J=9.5 Hz, 1H), 8.19-8.11(m, 2H), 8.11-8.06 (m, 2H), 8.05-7.96 (m, 2H), 7.66 (dt, J=8.5, 2.5 Hz,1H), 7.58-7.47 (m, 3H), 7.07 (d, J=8.6 Hz, 1H), 7.05 (d, J=1.9 Hz, 1H),6.96 (d, J=7.0 Hz, 1H), 6.91 (d, J=9.4 Hz, 1H), 6.53 (s, 1H), 4.97 (ddd,J=12.9, 5.4, 2.3 Hz, 1H), 3.63 (t, J=6.6 Hz, 2H), 3.56 (t, J=5.5 Hz,2H), 3.48-3.37 (m, 2H), 3.37-3.29 (m, 2H), 3.29-3.20 (m, 1H), 2.83-2.72(m, 1H), 2.72-2.58 (m, 2H), 2.54-2.47 (m, 2H), 2.34-2.25 (m, 1H),1.96-1.87 (m, 1H). LCMS: m/z 923.3 [M+1].

Example 2: Synthesis of2-(2,6-dioxopiperidin-3-yl)-4-((2-(2-(2-(3-(4-(4-(9-(isoquinolin-7-yl)-2-oxobenzo[h][1,6]naphthyridin-1(2H)-yl)-2-(trifluoromethyl)phenyl)piperazin-1-yl)-3-oxopropoxy)ethoxy)ethoxy)ethyl)amino)isoindoline-1,3-dione(I-2)

To a solution of9-(isoquinolin-7-yl)-1-(4-(piperazin-1-yl)-3-(trifluoromethyl)phenyl)benzo[h][1,6]naphthyridin-2(1H)-one (8.5 mg, 0.0154 mmol) and3-(2-(2-(2-((2-(2,6-dioxopiperidin-3-yl)-1,3-dioxoisoindolin-4-yl)amino)ethoxy)ethoxy)ethoxy)propanoicacid (10 mg, 0.0154 mmol) in DMF (0.5 mL) were added EDC (3.5 mg, 0.0185mmol), HOBT (2.7 mg, 0.02 mmol), and TEA (7.8 mg, 11 μL, 0.077 mmol).The mixture was stirred at rt overnight. The mixture was purified byreverse phase HPLC (0-100% MeOH in H₂O) to give compound I-2 as a yellowsolid (7.2 mg, 46%). ¹H NMR (500 MHz, DMSO-d₆) δ 11.02 (s, 1H), 9.45 (s,1H), 9.16 (s, 1H), 8.56 (d, J=6.0 Hz, 1H), 8.29 (d, J=9.5 Hz, 1H),8.19-8.11 (m, 3H), 8.09 (dd, J=8.7, 1.9 Hz, 1H), 8.03 (d, J=8.6 Hz, 1H),7.99 (d, J=2.4 Hz, 1H), 7.67 (dd, J=8.5, 2.5 Hz, 1H), 7.56 (d, J=8.6 Hz,1H), 7.53 (dd, J=8.6, 1.9 Hz, 1H), 7.48 (dd, J=8.6, 7.1 Hz, 1H),7.06-7.02 (m, 2H), 6.94 (d, J=7.0 Hz, 1H), 6.91 (d, J=9.4 Hz, 1H), 6.51(s, 1H), 4.98 (dd, J=12.7, 5.5 Hz, 1H), 3.60-3.50 (m, 3H), 3.50-3.40 (m,7H), 3.40-3.28 (m, 2H), 3.28-3.19 (m, 1H), 2.81 (ddd, J=16.8, 13.7, 5.4Hz, 1H), 2.73-2.65 (m, 1H), 2.65-2.58 (m, 1H), 2.55-2.51 (m, 1H),2.51-2.45 (m, 2H), 2.32-2.24 (m, 0H), 1.99-1.88 (m, 1H). LCMS: m/z1011.4 [M+1].

Example 3: Synthesis of2-(2,6-dioxopiperidin-3-yl)-4-((18-(4-(4-(9-(isoquinolin-7-yl)-2-oxobenzo[h][1,6]naphthyridin-1(2H)-yl)-2-(trifluoromethyl)phenyl)piperazin-1-yl)-18-oxo-3,6,9,12,15-pentaoxaoctadecyl)amino)isoindoline-1,3-dione(I-3)

To a solution of9-(isoquinolin-7-yl)-1-(4-(piperazin-1-yl)-3-(trifluoromethyl)phenyl)benzo[h][1,6]naphthyridin-2(1H)-one (8.3 mg, 0.015 mmol) and1-((2-(2,6-dioxopiperidin-3-yl)-1,3-dioxoisoindolin-4-yl)amino)-3,6,9,12,15-pentaoxaoctadecan-18-oicacid (11 mg, 0.015 mmol) in DMF (0.5 mL) were added EDC (3.5 mg, 0.018mmol), HOBT (2.6 mg, 0.02 mmol), and TEA (7.6 mg, 10 μl, 0.075 mmol).The mixture was stirred at rt overnight. The mixture was purified byreverse phase HPLC (0-100% MeOH in H₂O) to give compound I-3 as a yellowsolid (7.7 mg, 47%). ¹H NMR (500 MHz, DMSO-d₆) δ 11.02 (s, 1H), 9.48 (s,1H), 9.17 (s, 1H), 8.58 (d, J=6.0 Hz, 1H), 8.29 (d, J=9.5 Hz, 1H),8.21-8.13 (m, 3H), 8.09 (dd, J=8.7, 1.9 Hz, 1H), 8.06 (d, J=8.6 Hz, 1H),7.99 (d, J=2.5 Hz, 1H), 7.67 (dd, J=8.5, 2.4 Hz, 1H), 7.59-7.52 (m, 2H),7.49 (dd, J=8.6, 7.1 Hz, 1H), 7.05 (dd, J=5.3, 3.3 Hz, 2H), 6.95 (d,J=6.9 Hz, 1H), 6.91 (d, J=9.4 Hz, 1H), 6.52 (s, 1H), 4.98 (dd, J=12.7,5.4 Hz, 1H), 3.61-3.50 (m, 4H), 3.50-3.20 (m, 17H), 2.81 (ddd, J=16.9,13.7, 5.4 Hz, 1H), 2.74-2.66 (m, 1H), 2.65-2.57 (m, 1H), 2.55-2.51 (m,1H), 2.51-2.46 (m, 2H), 2.34-2.24 (m, 1H), 2.00-1.88 (m, 1H). LCMS: m/z1099.4 [M+1].

Example 4: Synthesis of(2R,4R)-1-((R)-2-(4-(4-(4-(5-chloro-4-(1-oxo-2,8-diazaspiro[4.5]decan-8-yl)pyridin-3-yl)phenyl)-1H-pyrazol-1-yl)butanamido)-3,3-dimethylbutanoyl)-4-hydroxy-N-(4-(4-methylthiazol-5-yl)benzyl)pyrrolidine-2-carboxamide(I-4)

Step 1: Synthesis of8-(3-(4-(1H-pyrazol-4-yl)phenyl)-5-chloropyridin-4-yl)-2,8-diazaspiro[4.5]decan-1-one

3-Bromo-4,5-dichloropyridine (454 mg, 2 mmol),2,8-diazaspiro[4.5]decan-1-one (381 mg, 2 mmol), and DIPEA (516 mg, 0.7mL, 4 mmol) were mixed in 5 mL of NMP. The mixture was heated to 120° C.and kept stirring overnight. The mixture was then cooled to roomtemperature and extracted with chloroform and isopropanol (V/V=4/1),dried over anhydrous Na₂SO₄ and concentrated. The crude was purified bycolumn chromatography on silica gel (0-10% MeOH in DCM) to give8-(3-bromo-5-chloropyridin-4-yl)-2,8-diazaspiro[4.5]decan-1-one (516 mg,75%). LCMS: m/z 344.0 [M+1].

To a sealed tube were added8-(3-bromo-5-chloropyridin-4-yl)-2,8-diazaspiro[4.5]decan-1-one (500 mg,1.45 mmol),4-(4-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)phenyl)-1H-pyrazole(783 mg, 2.9 mmol), Cs₂CO₃ (1.43 g, 4.35 mmol), Dioxane (5.4 mL) and H₂O(0.6 mL). The mixture was degassed and Pd(dppf)Cl₂ (118 mg, 0.15 mmol)was added. After addition, the mixture was heated to 110° C. and keptstirring overnight. The mixture was then cooled to room temperature andextracted with chloroform and isopropanol (V/V=4/1), dried overanhydrous Na₂SO₄ and concentrated. The crude was purified by columnchromatography on silica gel (0-10% MeOH in DCM) to give8-(3-(4-(1H-pyrazol-4-yl)phenyl)-5-chloropyridin-4-yl)-2,8-diazaspiro[4.5]decan-1-oneas a white solid (473.5 mg, 80%). LCMS: m/z 408.2 [M+1].

Step 2: Synthesis of(2R,4R)-1-((R)-2-(4-(4-(4-(5-chloro-4-(1-oxo-2,8-diazaspiro[4.5]decan-8-yl)pyridin-3-yl)phenyl)-1H-pyrazol-1-yl)butanamido)-3,3-dimethylbutanoyl)-4-hydroxy-N-(4-(4-methylthiazol-5-yl)benzyl)pyrrolidine-2-carboxamide(I-4)

To a solution of8-(3-(4-(1H-pyrazol-4-yl)phenyl)-5-chloropyridin-4-yl)-2,8-diazaspiro[4.5]decan-1-one(18.4 mg, 0.045 mmol) in Acetone (0.5 mL) was added tert-butyl4-bromobutanoate (15.1 mg, 0.068 mmol), followed by K₂CO₃ (12.3 mg, 0.09mmol) and KI (11.3 mg, 0.068 mmol). The mixture was then heated toreflux and kept stirring overnight. The mixture was diluted with EtOAcand H₂O, extracted, and washed with brine. The organic layer was driedover anhydrous Na₂SO₄ and concentrated. The crude was purified by columnchromatography on silica gel (0-10% MeOH in DCM) to give t-butyl esteras a gray solid (23.5 mg, 95%). LCMS: m/z 550.3 [M+1].

To a solution of the t-butyl ester (22 mg, 0.04 mmol) in DCM (0.5 mL)was added TFA (0.5 mL) and stirred at rt for 2 h. The mixture was thenconcentrated and dissolved in DMF (0.5 mL).(2S,4R)-1-((S)-2-amino-3,3-dimethylbutanoyl)-4-hydroxy-N-(4-(4-methylthiazol-5-yl)benzyl)pyrrolidine-2-carboxamide(17.2 mg, 0.04 mmol) was added, followed by EDCI (8.7 mg, 0.046 mmol),HOBT (6.6 mg, 0.049 mmol), and TEA (19 mg, 26 L, 0.19 mmol). The mixturewas stirred at rt overnight. The mixture was filtered and purified byreverse phase HPLC (0-100% MeOH in H₂O) to give compound I-4 as a yellowsolid (16.3 mg, 45% over two steps). ¹H NMR (500 MHz, DMSO-d₆) δ 8.97(s, 1H), 8.61-8.53 (m, 2H), 8.26 (d, J=3.9 Hz, 2H), 7.98 (d, J=13.0 Hz,2H), 7.75-7.69 (m, 3H), 7.53 (s, 1H), 7.42 (d, J=8.3 Hz, 2H), 7.37 (d,J=8.3 Hz, 2H), 7.33 (d, J=8.1 Hz, 2H), 4.55 (d, J=9.2 Hz, 1H), 4.48-4.40(m, 2H), 4.36 (dq, J=6.4, 3.2 Hz, 2H), 4.21 (dd, J=15.9, 5.5 Hz, 1H),4.12 (t, J=6.9 Hz, 2H), 3.68 (d, J=3.4 Hz, 2H), 3.11 (q, J=7.9, 6.7 Hz,4H), 2.75-2.65 (m, 2H), 2.43 (s, 3H), 2.03 (ddd, J=14.3, 11.3, 6.8 Hz,4H), 1.91 (ddd, J=12.9, 8.6, 4.6 Hz, 1H), 1.83 (t, J-=6.8 Hz, 2H), 1.72(td, J=12.5, 4.2 Hz, 2H), 1.27 (d, J=12.8 Hz, 2H), 0.95 (s, 9H). LCMS:m/z 906.4 [M+1].

Example 5: Synthesis of(2S,4R)-1-((S)-20-tert-butyl-1-(4-(4-(5-chloro-4-(1-oxo-2,8-diazaspiro[4.5]decan-8-yl)pyridin-3-yl)phenyl)-1H-pyrazol-1-yl)-18-oxo-3,6,9,12,15-pentaoxa-19-azahenicosane)-4-hydroxy-N-(4-(4-methylthiazol-5-yl)benzyl)pyrrolidine-2-carboxamide(I-5)

To a solution of8-(3-(4-(1H-pyrazol-4-yl)phenyl)-5-chloropyridin-4-yl)-2,8-diazaspiro[4.5]decan-1-one(18.4 mg, 0.045 mmol) in Acetone (0.5 mL) was added tert-butyl1-bromo-3,6,9,12,15-pentaoxaoctadecan-18-oate (30 mg, 0.068 mmol),followed by K₂CO₃ (12.3 mg, 0.09 mmol) and KI (11.3 mg, 0.068 mmol). Themixture was then heated to reflux and kept stirring overnight. Themixture was diluted with EtOAc and H₂O, extracted, and washed withbrine. The organic layer was dried over anhydrous Na₂SO₄ andconcentrated. The crude was purified by column chromatography on silicagel (0-10% MeOH in DCM) to give t-butyl ester (30.6 mg, 90%). LCMS: m/z756.4 [M+1].

To a solution of the t-butyl ester (30 mg, 0.04 mmol) in DCM (0.5 mL)was added TFA (0.5 mL) and stirred at rt for 2 h. The mixture was thenconcentrated and dissolved in DMF (0.5 mL).(2S,4R)-1-((S)-2-amino-3,3-dimethylbutanoyl)-4-hydroxy-N-(4-(4-methylthiazol-5-yl)benzyl)pyrrolidine-2-carboxamide(17.2 mg, 0.04 mmol) was added, followed by EDCI (8.7 mg, 0.046 mmol),HOBT (6.6 mg, 0.049 mmol), and TEA (19 mg, 26 μL, 0.19 mmol). Themixture was stirred at rt overnight. The mixture was filtered andpurified by reverse phase HPLC (0-100% MeOH in H₂O) to give compound I-5as a yellow solid (15.6 mg, 35% over two steps). ¹H NMR (500 MHz,DMSO-d₆) δ 8.95 (d, J=3.4 Hz, 1H), 8.60 (s, 1H), 8.52 (t, J=6.1 Hz, 1H),8.29 (d, J=18.7 Hz, 1H), 8.22 (d, J=7.1 Hz, 1H), 7.98-7.92 (m, 1H), 7.87(d, J=9.3 Hz, 1H), 7.67 (d, J=8.1 Hz, 2H), 7.50 (s, 1H), 7.41-7.29 (m,6H), 4.51 (d, J=9.3 Hz, 1H), 4.42-4.35 (m, 2H), 4.31 (d, J=4.2 Hz, 1H),4.25 (t, J=5.4 Hz, 2H), 4.17 (dd, J=15.9, 5.5 Hz, 1H), 3.77 (t, J=5.4Hz, 2H), 3.63 (dd, J=10.5, 4.1 Hz, 1H), 3.60-3.56 (m, 1H), 3.49 (dd,J=5.8, 2.8 Hz, 2H), 3.45-3.40 (m, 8H), 3.14 (d, J=12.9 Hz, 2H), 3.07 (t,J=6.8 Hz, 2H), 2.69 (t, J=12.1 Hz, 2H), 2.46 (p, J=1.9 Hz, 8H), 2.40 (s,3H), 2.34-2.26 (m, 1H), 1.99 (t, J=10.5 Hz, 1H), 1.86 (ddd, J=13.0, 8.6,4.8 Hz, 2H), 1.81 (t, J=6.8 Hz, 2H), 1.69 (td, J=12.4, 12.0, 4.0 Hz,2H), 1.26 (d, J=13.0 Hz, 2H), 0.89 (s, 91). LCMS: m/z 1112.5 [M+1].

Example 6: Synthesis of4-(4-(4-(4-(5-chloro-4-(1-oxo-2,8-diazaspiro[4.5]decan-8-yl)pyridin-3-yl)phenyl)-1H-pyrazol-1-yl)butylamino)-2-(2,6-dioxopiperidin-3-yl)isoindoline-1,3-dione(I-6)

To a solution of8-(3-(4-(1H-pyrazol-4-yl)phenyl)-5-chloropyridin-4-yl)-2,8-diazaspiro[4.5]decan-1-one(18.4 mg, 0.045 mmol) in Acetone (0.5 mL) was added tert-butyl4-bromobutylcarbamate (17.2 mg, 0.068 mmol), followed by K₂CO₃ (12.3 mg,0.09 mmol) and KI (11.3 mg, 0.068 mmol). The mixture was then heated toreflux and kept stirring overnight. The mixture was diluted with EtOAcand H₂O, extracted, and washed with brine. The organic layer was driedover anhydrous Na₂SO₄ and concentrated. The crude was purified by columnchromatography on silica gel (0-10% MeOH in DCM) to give Boc protectedamine (24.7 mg, 95%). LCMS: m/z 578.2 [M+1].

To a solution of the Boc protected amine (23 mg, 0.04 mmol) in DCM (0.5mL) was added TFA (0.5 mL) and stirred at rt for 2 h. The mixture wasthen concentrated and dissolved in DMF (0.5 mL).2-(2,6-dioxopiperidin-3-yl)-4-fluoroisoindoline-1,3-dione (11.1 mg, 0.04mmol) was added, followed by EDCI (8.7 mg, 0.046 mmol), HOBT (6.6 mg,0.049 mmol), and TEA (19 mg, 26 μL, 0.19 mmol). The mixture was stirredat rt overnight. The mixture was filtered and purified by reverse phaseHPLC (0-100% MeOH in H₂O) to give compound I-6 as a yellow solid (11.2mg, 38% over two steps). ¹H NMR (500 MHz, DMSO-d₆) δ 11.08 (s, 1H), 8.55(s, 1H), 8.28 (s, 1H), 8.26 (s, 1H), 7.97 (s, 1H), 7.70 (s, 1H), 7.68(d, J=2.1 Hz, 1H), 7.56 (dd, J=8.6, 7.1 Hz, 1H), 7.53 (s, 1H), 7.34 (s,1H), 7.33 (s, 11H), 7.10 (d, J=8.6 Hz, 1H), 7.00 (d, J=7.0 Hz, 1H), 6.62(s, 1H), 5.04 (dd, J=12.7, 5.4 Hz, 1H), 4.19 (t, J=6.9 Hz, 2H), 3.34 (s,2H), 3.11 (td, J=6.5, 3.3 Hz, 4H), 2.75-2.65 (m, 2H), 2.62-2.55 (m, 1H),2.54 (s, 2H), 2.06-1.97 (m, 1H), 1.90 (p, J=7.0 Hz, 2H), 1.84 (t, J=6.8Hz, 2H), 1.72 (td, J=12.4, 4.2 Hz, 2H), 1.62-1.51 (m, 2H), 1.28 (d,J=13.1 Hz, 2H). LCMS: m/z 735.3 [M+1].

Example 7:2-(4-(4-(5-chloro-4-(1-oxo-2,8-diazaspiro[4.5]decan-8-yl)pyridin-3-yl)phenyl)-1H-pyrazol-1-yl)-N-(2-(2-(2-(2-(2-(2,6-dioxopiperidin-3-yl)-1,3-dioxoisoindolin-4-ylamino)ethoxy)ethoxy)ethoxy)ethyl)acetamide(I-7)

To a solution of8-(3-(4-(1H-pyrazol-4-yl)phenyl)-5-chloropyridin-4-yl)-2,8-diazaspiro[4.5]decan-1-one(18.4 mg, 0.045 mmol) in Acetone (0.5 mL) was added tert-butyl2-bromoacetate (13.2 mg, 0.068 mmol), followed by K₂CO₃ (12.3 mg, 0.09mmol) and KI (11.3 mg, 0.068 mmol). The mixture was then heated toreflux and kept stirring overnight. The mixture was diluted with EtOAcand H₂O, extracted, and washed with brine. The organic layer was driedover anhydrous Na₂SO₄ and concentrated. The crude was purified by columnchromatography on silica gel (0-10% MeOH in DCM) to give t-butyl ester(22.3 mg, 95%). LCMS: m/z 522.2 [M+1].

To a solution of the t-butyl ester (20.8 mg, 0.04 mmol) in DCM (0.5 mL)was added TFA (0.5 mL) and stirred at rt for 2 h. The mixture was thenconcentrated and dissolved in DMF (0.5 mL).4-(2-(2-(2-(2-aminoethoxy)ethoxy)ethoxy)ethylamino)-2-(2,6-dioxopiperidin-3-yl)isoindoline-1,3-dione(17.9 mg, 0.04 mmol) was added, followed by EDCI (8.7 mg, 0.046 mmol),HOBT (6.6 mg, 0.049 mmol), and TEA (19 mg, 26 μL, 0.19 mmol). Themixture was stirred at rt overnight. The mixture was filtered andpurified by reverse phase HPLC (0-100% MeOH 5 in H₂O) to give compoundI-7 as a yellow solid (11.8 mg, 33% over two steps). ¹H NMR (500 MHz,DMSO-d₆) δ 11.09 (s, 1H), 8.57 (s, 1H), 8.28 (s, 1H), 8.21 (d, J=9.4 Hz,1H), 7.98 (s, 1H), 7.70 (dd, J=7.6, 4.6 Hz, 2H), 7.60-7.50 (m, 2H), 7.35(d, J=8.0 Hz, 2), 7.14 (d, J=8.6 Hz, 1H), 7.04 (d, J=7.0 Hz, 1H), 6.59(d, J=15.1 Hz, 2H), 5.05 (dd, J=12.7, 5.4 Hz, 1H), 4.83 (s, 2H), 3.62(t, J=5.4 Hz, 2H), 3.59-3.41 (m, 9H), 3.25 (q, J=5.6 Hz, 2H), 3.11 (q,J=6.6, 6.1 Hz, 3H), 2.94-2.82 (m, 2H), 2.69 (t, J=12.1 Hz, 2H), 2.58(dt, J=16.9, 2.9 Hz, 1H), 2.02 (ddt, J=12.5, 5.6, 2.9 Hz, 4H), 1.84 (t,J=6.8 Hz, 2H), 1.72 (td, J=12.5, 4.2 Hz, 2H), 1.28 (d, J=13.0 Hz, 2H).LCMS: m/z 896.3 [M+1].

Example 8:4-(4-(4-(5-chloro-4-(1-oxo-2,8-diazaspiro[4.5]decan-8-yl)pyridin-3-yl)phenyl)-1H-pyrazol-1-yl)-N-(2-(2-(2-(2-(2-(2,6-dioxopiperidin-3-yl)-1,3-dioxoisoindolin-4-ylamino)ethoxy)ethoxy)ethoxy)ethyl)butanamide(I-8)

To a solution of8-(3-(4-(1H-pyrazol-4-yl)phenyl)-5-chloropyridin-4-yl)-2,8-diazaspiro[4.5]decan-1-one(18.4 mg, 0.045 mmol) in Acetone (0.5 mL) was added tert-butyl4-bromobutanoate (15.1 mg, 0.068 mmol), followed by K₂CO₃ (12.3 mg, 0.09mmol) and KI (11.3 mg, 0.068 mmol). The mixture was then heated toreflux and kept stirring overnight. The mixture was diluted with EtOAcand H₂O, extracted, and washed with brine. The organic layer was driedover anhydrous Na₂SO₄ and concentrated. The crude was purified by columnchromatography on silica gel (0-10% MeOH in DCM) to give t-butyl ester(23.5 mg, 95%). LCMS: m/z 550.3 [M+1].

To a solution of the t-butyl ester (22 mg, 0.04 mmol) in DCM (0.5 mL)was added TFA (0.5 mL) and stirred at rt for 2 h. The mixture was thenconcentrated and dissolved in DMF (0.5 mL).4-(2-(2-(2-(2-aminoethoxy)ethoxy)ethoxy)ethylamino)-2-(2,6-dioxopiperidin-3-yl)isoindoline-1,3-dione(17.9 mg, 0.04 mmol) was added, followed by EDCI (8.7 mg, 0.046 mmol),HOBT (6.6 mg, 0.049 mmol), and TEA (19 mg, 26 μL, 0.19 mmol). Themixture was stirred at rt overnight. The mixture was filtered andpurified by reverse phase HPLC (0-100% MeOH in H₂O) to give compound I-8as a yellow solid (11.1 mg, 30% over two steps). ¹H NMR (500 MHz,DMSO-d₆) δ 11.09 (s, 1H), 8.56 (s, 1H), 8.25 (d, J=6.2 Hz, 2H), 7.96 (s,1H), 7.89 (t, J=5.6 Hz, 1H), 7.74-7.67 (m, 2H), 7.57 (dd, J=8.6, 7.1 Hz,1H), 7.53 (d, J=2.8 Hz, 1H), 7.38-7.32 (m, 2H), 7.13 (d, J=8.6 Hz, 1H),7.03 (d, J=7.0 Hz, 1H), 6.58 (d, J=10.4 Hz, 1H), 5.05 (dd, J=12.8, 5.5Hz, 1H), 4.12 (t, J=6.8 Hz, 2H), 3.60 (t, J=5.4 Hz, 2H), 3.57-3.42 (m,10H), 3.39 (t, J=5.9 Hz, 2H), 3.19 (q, J=5.8 Hz, 2H), 3.15-3.06 (m, 4H),2.76-2.65 (m, 2H), 2.62-2.55 (m, 1H), 2.54 (s, 1H), 2.13-2.06 (m, 2H),2.06-1.97 (m, 4H), 1.84 (t, 0.1=6.8 Hz, 2H), 1.72 (td, J=12.5, 4.1 Hz,2H), 1.28 (d, J=12.9 Hz, 2H). LCMS: m/z 924.4 [M+1].

Example 9: Biochemical Studies

Cell Viability Assay

Molt4 wild-type or cereblon null cells were treated with a titration ofa bifunctional compound of the invention (i.e., Compound I-1, CompoundI-2, or Compound I-3) and allowed to grow for 72 hours. Cells wereassayed using celltiter glo (Promega) to determine cell viability bymeasuring the amount of ATP present, which is an indicator of cellmetabolic activity. Results are graphed as relative luminescent values.The results are shown in FIG. 1A and FIG. 1B.

Enzyme Degradation Assay

The indicated cells were treated with vehicle control or thebifunctional compounds of the application, at a single or variousconcentrations, alone or in combination with bortezomib, an agent thatblocks proteasomal degradation. After treatment, cells were washed withPBS and lysed in lysis buffer and then cleared by centrifugation.Protein concentrations were determined by BCA assay, and equal amountsof protein were loaded onto gels to perform Western blots. Gels weretransferred to nitrocellulose, blocked with 5% milk in TBS-T, and probedfor CDK8, mTOR, S727-STAT1, and Actin. Antibody binding was detectedusing near-infrared-dye-conjugated secondary antibodies. The results areshown in FIG. 2 , FIG. 3A and FIG. 3B.

Western Blotting on CDK8 Cells were treated with the indicated compoundsat the indicated concentrations for the indicated amount of time. Cellswere then lysed in M-PER buffer (Thermo Scientific) containingprotease/phosphatase inhibitor cocktail (Roche). Protein concentrationwas measured using a BCA assay (Pierce). Equivalent amounts of eachsamples were loaded on 4-12% Bis-Tris gels (Invitrogen), transferred tonitrocellulose membranes, and immunoblotted with antibodies against CDK8and Actin (Cell Signaling). IRDye® 800-labeled goat anti-rabbit IgG andIRDye® 680-labeled goat anti-mouse IgG (LI-COR) secondary antibodieswere purchased for LI-COR, and membranes were detected on an Odysseydetection system (LI-COR Biosciences). The results are shown in FIG. 2and FIG. 4A-4C.

EQUIVALENTS

Those skilled in the art will recognize, or be able to ascertain usingno more than routine experimentation, many equivalents to the specificembodiments and methods described herein. Such equivalents are intendedto be encompassed by the scope of the present application.

All patents, patent applications, and literature references cited hereinare hereby expressly incorporated by reference.

The invention claimed is:
 1. A bifunctional compound of Formula X:TL-L-D  (X), wherein: the Targeting Ligand (TL) is of Formula TL-I:

wherein: A is

X is N or CH; X₂ is N or CH; X₃ is N or CH; X₄ is N or CH; each R₁ isindependently (C₁-C₄) alkyl or (C₁-C₄) haloalkyl; each R₂ isindependently (C₁-C₄) alkyl, (C₁-C₄) haloalkyl, (C₁-C₄) alkoxy, (C₁-C₄)haloalkoxy, halogen, OH, or NH₂; each R₃ is independently (C₁-C₄) alkyl,(C₁-C₄) haloalkyl, (C₁-C₄) alkoxy, (C₁-C₄) haloalkoxy, halogen, OH, orNH₂; each R₄ is independently (C₁-C₄) alkyl, (C₁-C₄) haloalkyl, (C₁-C₄)alkoxy, (C₁-C₄) haloalkoxy, halogen, OH, or NH₂; and t, n, o, and r areeach independently 0, 1, 2, or 3; wherein the Targeting Ligand is bondedto the Linker via the

next to

the Linker (L) is a group that covalently binds to the Targeting Ligandand the Degron; and the Degron (D) is of Formula D2:

wherein: each R₁₇ is independently (C₁-C₃) alkyl; q′ is 0, 1, 2, 3, or4; and R₁₈ is H or (C₁-C₃) alkyl, wherein the Degron is covalentlybonded to the Linker via the

or a stereoisomer or pharmaceutically acceptable salt thereof.
 2. Thebifunctional compound of claim 1, wherein A is


3. The bifunctional compound of claim 1, wherein X is N.
 4. Thebifunctional compound of claim 1, wherein X₂ is N.
 5. The bifunctionalcompound of claim 1, wherein X₃ is N.
 6. The bifunctional compound ofclaim 1, wherein t is
 0. 7. The bifunctional compound of claim 1,wherein o is
 0. 8. The bifunctional compound of claim 1, wherein n is 0.9. The bifunctional compound of claim 1, wherein r is 1 and R₄ is(C₁-C₄) haloalkyl.
 10. The bifunctional compound of claim 1, wherein theTargeting Ligand is of Formula TL-Ia, TL-Ib, TL-Ic, TL-Id, TL-Ie, orTL-If:


11. The bifunctional compound of claim 1, wherein the Linker is ofFormula L0:

or a stereoisomer thereof, wherein p1 is an integer selected from 0 to12; p2 is an integer selected from 0 to 12; p3 is an integer selectedfrom 1 to 6; each W is independently absent, CH₂, O, S, NH, or NR₁₉; Z₁is absent, C(O), (CH₂)_(j)C(O)NH, CH₂, O, NH, or NR₁₉; each R₁₉ isindependently C₁-C₃ alkyl; j is 1, 2, or 3; and Q is absent orNHC(O)CH₂, wherein the Linker is covalently bonded to the Degron via the

next to Q, and covalently bonded to the Targeting Ligand via the

next to Z₁.
 12. The bifunctional compound of claim 11, wherein theLinker is selected from:


13. The bifunctional compound of claim 1, wherein R₁₈ is methyl.
 14. Thebifunctional compound of claim 1, wherein the Degron is of Formula D2aor D2b:


15. A pharmaceutical composition comprising a therapeutically effectiveamount of the bifunctional compound of claim 1, or a stereoisomer orpharmaceutically acceptable salt thereof, and a pharmaceuticallyacceptable carrier.
 16. A method of treating cancer, wherein the canceris leukemia or lymphoma, comprising administering to a subject in needthereof an effective amount of the bifunctional compound orpharmaceutically acceptable salt or stereoisomer of claim 1.